Yeast-based assay

ABSTRACT

The application discloses  Sz. pombe  yeast cells which have been modified so that a F-Protein Coupled Receptor (GPCR)-regulated signalling pathway is derepressed during the mitotic phase of cell growth. Isolated nucleic acid molecules encoding constructs used to make the yeast cells, uses of the cells and nucleic acid molecules to study GPCR pathways and to isolate compounds which effect in such pathways are also disclosed.

[0001] The application relates to modified yeast cells which may be used to study the activity of G-protein coupled receptors (GPCRs). The yeast cells used are Schizosaccharomyces pombe (Sz. pombe) containing a reporter gene-promoter construct. The invention also relates to isolated nucleic acid encoding the reporter gene-promoter construct and to uses of the yeast cells and nucleic acid molecules in assays.

[0002] GPCRs are an important class of receptors in all eukaryotic organisms, including mammals and yeast, and are responsible for conveying hormonal and sensory signals to the cell machinery (reviewed in Baldwin, 1994). Such receptors have a common structure comprising 7-transmembrane domains with an extracellular N-terminus and C-terminal cytoplasmic tail. GPCRs are usually coupled to a heterotrimeric G protein composed of Gα, Gβ and Gγ subunits. Binding of a ligand to the receptor stimulates a change in the G protein where guanosine diphosphate (GDP) bound to the Gα subunit is exchanged for guanosine triphosphate (GTP). Accompanying conformational changes result in the dissociation of Gα-GTP from the Gβγ dimer, either of which can modulate the activity of effector proteins to bring about changes in cell behaviour.

[0003] GPCRs control the physiology of all major organ systems and have been important targets for therapeutic and diagnostic advances, providing clinically successful drugs in nearly all the major pharmaceutical markets. Many of the 200 or so well characterised GPCRs are associated with at least one drug and about 60% of commercially available drugs act on GPCRs, providing some $27 billion in annual sales world-wide. There are another 100 or so GPCRs for which ligands have not yet been identified. These so called ‘orphan' receptors are likely to include many that will become important drug targets. Analysis of the human genome indicates that there are probably another 500 orphan GPCRs that will need to be characterised. There is therefore considerable interest in developing drug leads targeted at the GPCRs.

[0004] One approach to the identification of new drugs is the development of high throughput screens (HTS) for GPCRs. In most cases, the target GPCR is expressed in a host system such that activation of the receptor leads to a change in cell behaviour. Screening can then identify drug leads that either mimic the action of the natural ligand (agonists) or block the receptor (antagonists). All eukaryotic cells contain GPCRs and each can be adapted for HTS but it is not always practical to do this and most screens use a limited range of host systems. These include mammalian cells, frog melanocytes, insect cells and yeast Each system has its advantages and disadvantages. For example, mammalian cells might seem the obvious choice for studying human GPCRs but they are difficult and expensive to work with and screens are often complicated by the inherent presence of receptors closely related to the GPCR being studied. The presence of related receptors can also complicate screens involving frog melanocytes and insect cells. These problems do not apply to yeast and many have turned to using this relatively simple cell as a surrogate host for screening human GPCRs.

[0005] G-protein coupled receptors are known in yeast. Accordingly, yeast, such as Saccharomyces cerevisiae (S. cerevisiae) have been used to study GPCR-regulated signalling systems. Yeast cells are particularly advantageous because they have the ability to be easily manipulated, at low cost and with high levels of production. Unlike bacteria, yeast has the potential to perform eukaryotic post-translational modifications that may affect receptor function (Reiländer and Weib, 1998). The mechanism of transcriptional activation in yeast and higher eukaryotes may be very similar. For example, yeast upstream activation sites (UAS) and some transcriptional activators have been found to have very similar activity to that of their mammalian equivalents (Jones et al., 1988).

[0006] Most work carried out on yeast systems has been on S. cerevisiae. There are many reports describing the coupling of exogenous GPCRs to the intracellular signalling machinery in S. cerevisiae. These include the human β₂-adrenergic (King et al., 1990), rat somatostatin (Price et al., 1995; Bass et al., 1996), rat adenosine A_(2A) (Price et al., 1996), human growth hormone releasing hormone (Kajkowski et al., 1997), human lysophosphatidic acid (Erickson et al., 1998), human formyl peptide receptor like-1 (Klein et al., 1998), human C5a chemoattractant (Klein et al., 1998; Baranski et al., 1999), mushroom pheromone (Olesnicky et al., 1999), human somatostatin SST₂ (Brown et al., 2000), human somatostatin SST₅ (Brown et al., 2000), human serotonin 5-HT_(1A) (Brown et al., 2000), human serotonin 5-HT_(1D) (Brown et al., 2000), human melatonin ML_(1B) (Brown et al., 2000), human purinergic P2Y₁ (Brown et al., 2000), human purinergic P2Y₂ (Brown et al., 2000), human adenosine A_(2B) (Brown et al., 2000), human UDP-glucose (Chambers et al., 2000), human protease-activated receptor (Swift et al., 2000), human muscarinic M₁ (Erlenbach et al., 2001), human muscarinic M₃ Erlenbach et al., 2001), human muscarinic M₅ (Erlenbach et al., 2001) and human vasopressin V₂ (Erlenbach et al., 2001).

[0007] However, not all GPCRs couple to the signalling machinery in S. cerevisiae. Receptors that fail to couple are not normally reported but as many as 40% of human GPCRs are not functional in S. cerevisiae.

[0008] The fission yeast Schizosaccharomyces pombe (Sz. pombe) is becoming popular as an alternative genetically tractable eukaryote which is not only phylogenetically distant from S. cerevisiae, but in several aspects of its cell and molecular biology seems to more closely resemble a higher eukaryotic cell (Reiländer and Weib, 1998; Allshire et al., 1987). Sz. pombe would therefore seem to provide an attractive alternative to the budding yeast. Unfortunately, all previously reported attempts to couple exogenous GPCRs to the signalling machinery in Sz. pombe have been unsuccessful. It appears that although the receptors are expressed they fail to couple to the intracellular signalling machinery in the yeast Examples include bacteriorhodopsin (Hildebrandt et al., 1993), human dopamine D_(2S) (Sander et al., 1994); human neurokinin NK2 (Arkinstall et al., 1995) and human β₂-adrenergic (Ficca et al., 1995).

[0009] This application describes how Sz. pombe may be manipulated, for example by way of modification, to allow the coupling of exogenous GPCRs to the intracellular signalling machinery and hence generate strains suitable for high throughput screening for agonists and antagonists that affect the activity of the exogenous receptors.

[0010] Fission yeasts, such as Sz. pombe, have two distinct growth cycles. Firstly, they have a normal vegetative or mitotic cycle in which haploid cells simply divide by fission. Secondly, they have a meiotic cycle. In the meiotic cycle, a yeast cell conjugates with a second yeast cell to form a diploid cell. The diploid cell then undergoes meiosis and sporulation to form four haploid spores. Such spores are very resilient and the meiotic cycle is usually triggered when environmental conditions for the yeast no longer support mitotic growth. For example, the meiotic cycle in Sz. pombe is usually triggered by nitrogen starvation.

[0011] Conjugation in Sz. pombe is controlled by the reciprocal action of diffusible mating pheromones. M cells (of mating type minus) release M-factor which prepares P cells (mating type plus) for mating, while P cells release P-factor which stimulates M cells for mating. Binding of the pheromones to their receptors on the surface of the target cell activates an intracellular signalling pathway which leads to changes in the pattern of gene transcription and prepares the cell for mating. Responses induced by the pheromones include G₁ arrest of the cell cycle, an increase in agglutination, and the elongation of the cell to form a shmoo. The M-receptor and P-receptor to which the M-factor and P-factor pheromones bind are examples of G-protein coupled receptors. On binding of the pheromone to the receptor, a Gα subunit is released. This has a positive role in signal transduction within the Sz. pombe cell, as indeed is the case in many mammalian cells. This contrasts with S. cerevisiae, in which the Gα subunit is a negative regulator. Accordingly, the Sz. pombe system can be thought to be more closely analogous to GPCRs in higher eukaryotes such as mammals.

[0012] It has now been realised that an appropriately modified Sz. pombe would be a good model for studying GPCRs for identifying components of GPCR pathways, for identifying mutants in the GPCR pathways and for identifying compounds which stimulate or inhibit GPCR-regulated signalling pathways.

[0013]Sz. pombe also appears to have greater cell wall permeability than S. cerevisiae. This may prove to be invaluable in the study of receptors with large or complex ligands.

[0014] A first aspect of the invention provides a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising:

[0015] (i) a G-Protein Coupled Receptor (GPCR)-regulated signalling pathway which is derepressed during the mitotic phase of cell growth;

[0016] (ii) a reporter system for reporting a signal mediated by the GPCR-regulated signalling pathway

[0017] and wherein:

[0018] (a) the GPCR is heterologous, and/or

[0019] (b) the reporter system comprises a reporter gene and a promoter, the reporter gene being operatively linked to the promoter, and the promoter being regulatable by the GPCR, at least one of the reporter genes and the promoter being heterologous.

[0020] Expression of some of the components of the GPCR-regulated signalling machinery is normally repressed in Sz. pombe during mitotic growth and it is necessary to remove this repression in order to study signalling during the mitotic phase of cell growth. Methods for derepressing the GPCR-regulated signalling machinery are discussed below.

[0021] It has been discovered that maintaining a derepressed GPCR-regulated signalling pathway during the mitotic phase of cell growth allows Sz. pombe to be used to study GPCRs. That is, the cell has one or more signalling components activated during the mitotic phase to enable, for example, the binding of a suitable ligand to the GPCR to increase or reduce transcription of a reporter gene.

[0022] The yeast cell will generally comprise one or more mutations to derepress the GPCR-regulated pathway in mitotic growth. A nutritional control pathway can be disrupted by mutation for this purpose.

[0023] It is normally necessary to starve Sz. pombe cells to induce them to mate and derepress the GPCR-regulated signalling pathway. The relatively high level of cytoplasmic cAMP that exists during mitotic growth is reduced as nutrients become limiting and this helps to trigger sexual development. Strains lacking adenylate cyclase (which converts ATP to cAMP) have no cytoplasmic cAMP but grow reasonably well. They are derepressed for sexual development and respond to mating pheromones during mitotic growth. Accordingly, preferably the yeast cell is adenylate cyclase deficient. More especially, the cyr1 gene, which encodes adenylate cyclase, is physically or functionally removed or disrupted, for example by insertion of a DNA sequence. The inserted DNA sequence may be anything convenient, but in a preferred embodiment of the invention the inserted DNA may comprise a reporter gene; the ura4 gene is one example, as will be discussed below. The cyr1 gene is discussed in detail in the article by John Davey and Olaf Nielsen (Davey and Nielsen, 1994). Other methods for bypassing the nutritional control of the signalling machinery are available and may be used to derepress the GPCR-regulated signalling pathway in cells of the invention. This could include mutation of any gene that has the effect of repressing sexual differentiation. Such genes include those encoding certain protein kinases repressing sexual differentiation, including the pat1 gene. The use of a mutation in this gene to bypass the nutritional control and derepress the GPCR-regulated signalling pathway has been described (Davey and Nielsen, 1994).

[0024] As indicated above, Davey and Nielsen, 1994 discloses the identification of mutants involved in sexual differentiation and pheromone response. A temperature-sensitive pat1 mutant (pat1-114) allows the arrest of mitotic growth in response to M-factor. A mutation in the adenylate cyclase gene (cyr1) was also studied. The authors indicated that cells containing such a mutation have a problem in that they become insensitive or adapted to the pheromone. The perceived problems identified by the authors of the paper, have, in contrast with the analogous situation in Saccharomyces cerevisiae, now been found not to present a practical difficulty in heterologous Sz. pombe systems: specifically, it has been found that Sz. pombe cells containing a mutant cyr1 and a heterologous GPCR or a suitable heterologous reporter gene do not have a serious problem with desensitisation.

[0025] The reporter system allows signal transduction to be measured in a variety of ways. For example, suitable reporter system includes the association or dissociation of signalling components (to include, for example, the association of proteins with stimulated GPCRs, the dissociation of Gα subunits from negative regulators such as the Gβγ subunits), the generation of second messengers (such as Ca²⁺ mobilisation, changes in cyclic AMP levels, GTP hydrolysis, phospholipid hydrolysis), the modification of signalling components (such as the phosphorylation of e.g. MAP kinases, MAP kinase kinases or MAP kinase kinase kinases) or altered transcription of a gene. Transcription of a gene can be measured directly (for example, mRNA expression may be detected by Northern blots) or indirectly (for example, the protein product may be measured by a characteristic stain or intrinsic activity).

[0026] Preferably the yeast comprises a nucleic acid molecule encoding a heterologous reporter gene, or an endogenous reporter gene, operatively linked to a promoter that is regulated by a GPCR-regulated signalling pathway. By operatively linked, we mean that the heterologous reporter gene, or the endogenous reporter gene, is linked to the promoter in such a way that the promoter is capable of directing transcription of the reporter gene.

[0027] The reporter gene may be any nucleic acid sequence encoding a detectable gene product. The gene product may be an untranslated RNA product such as mRNA or antisense RNA. Such untranslated RNA may be detected by techniques known in the art, such as PCR, Northern or Southern blots. Alternatively, the reporter gene may encode a polypeptide, such as protein or peptide, product. A polypeptide may be detected immunologically or by means of its biological activity. The reporter gene may be any known in the art. The reporter gene need not be a natural gene, and the term “gene” in this sense should not be taken to imply identity-with any natural gene. Reporter genes useful in the invention may be the same as certain natural genes, but may differ from them either in terms of non-coding sequences (for example one or more naturally occurring introns may be absent) or in terms of coding sequences.

[0028] The reporter gene may encode a protein that allows the yeast cell to be selected by, for example, a nutritional requirement. For example, the reporter gene may be the ura4 gene which encodes orotidine-5′-phosphate decarboxylase. The ura4 gene encodes an enzyme involved in the biosynthesis of uracil and offers both positive and negative selection. Only cells expressing ura4 are able to grow in the absence of uracil, where the appropriate yeast strain is used. Cells expressing ura4 die in the presence of 5-fluoro-orotic acid (FOA) as the ura4 gene product converts FOA into a toxic product. Cells not expressing ura4 can be maintained by adding uracil to the medium. The sensitivity of the selection process can be adjusted by using medium containing 6-azauracil, a competitive inhibitor of the ura4 gene product. The his3 gene (encodes imidazoleglycerol-phosphate dehydratase) is also suitable for use as a reporter gene that allows nutritional selection. Only cells expressing his3 are able to grow in the absence of histidine, where the appropriate yeast strain is used

[0029] The reporter gene may encode for a protein that allows the yeast to be used in a chromogenic assay. For example, the reporter may be the lacZ gene from Escherichia coli. This encodes the β-galactosidase enzyme. This catalyses the hydrolysis of β-galactoside sugars such as lactose. The enzymatic activity of the enzyme may be assayed with various specialised substrates, for example X-gal (5-bromo-4-chloro-3-indoyl-β-D-galactoside) or o-nitrophenyl-β-D-galactopyranoside, which allow reporter enzyme activity to be assayed using a spectrophotometer, fluorometer or a luminometer.

[0030] The gene encoding green fluorescent protein (GFP), which is known in the art, may also be used as a reporter gene.

[0031] The reporter gene may also encode a protein that is capable of inducing the cell, or an extract of a cell, to produce light. For example, the reporter gene may encode luciferase. The luciferase reporter genes are known in the art. They are usually derived from firefly (Photinous pyralis) or sea pansy (Renilla reniformis). The luciferase enzyme catalyses a reaction using D-luciferin and ATP in the presence of oxygen and Mg²⁺ resulting in light emission. The luciferase reaction is quantitated using a luminometer that measures light output. The assay may also include coenzyme A in the reaction that provides a longer, sustained light reaction with greater sensitivity.

[0032] An alternative form of enzyme that allows the production of light is aequorin, which is known in the art.

[0033] Most preferably, the reporter gene encodes β-lactamase. This reporter gene has certain advantages over, for example, lacZ. There is no background activity in mammalian cells or yeast cells, it is compact (29 kDa), it functions as a monomer (in comparison with lacZ which is a tetramer), and has good enzyme activity. This may use CCF2/AM, a FRET-based membrane permeable, intracellularly trapped fluorescent substrate. CCF2/AM has a 7-hydroxycoumarin linked to a fluorescein by a cephalosporin core. In the intact molecules, excitation of the coumarin results in efficient FRET to the fluorescein, resulting in green fluorescent Cleavage of the CCF2 by β-lactamase results in spatial separation of the two dyes, disrupting FRET and causing cells to change from green to blue when viewed using a fluorescent microscope. The retention of the cleaved product allows the blue colour to develop over time, giving a low detection limit of, for example, 50 enzyme molecules per cell. This results in the reporter gene being able to be assayed with high sensitivity. It also allows the ability to confirm results by visual inspection of the cells or the samples.

[0034] The nucleic acid molecule comprising the reporter gene under the control of the GPCR-regulated promoter may additionally comprise one or more additional regulatory elements, such as upstream activating sequences (UAS), termination sequences and/or secretory sequences known in the art. The secretory sequences may be used to ensure that the product of the reporter gene is secreted out of the yeast cell.

[0035] Preferably the promoter is regulatable by a yeast mating pheromone binding to its GPCR. The yeast mating pheromone may especially be P-factor pheromone. This is especially preferred because the P-factor pheromone is relatively easy to produce.

[0036] The promoter is preferably an endogenous Sz. pombe promoter which is regulated by the GPCR. However, it does not have to be endogenous. Certain heterologous promoters may be found to be so regulatable, or may be engineered to be, for example by inclusion of a TR-box motif as described by Aono, et al. (1994).

[0037] More preferably, the promoter is the sxa2 promoter, or a homologue or analogue thereof. By homologue or analogue we mean a promoter which may contain one or more changes to the nucleic acid sequence encoding the sxa2 promoter but which maintains the same functional activity as the sxa2 promoter. The sxa2 gene to which the sxa2 promoter is attached in wild-type cells, encodes a carboxypeptidase that, in wild-type cells, inactivates P-factor by removal of the C-terminal leucine residue (Ladds et al., 1996). Use of the sxa2 promoter for construction of a GPCR-regulated reporter is advantageous because the promoter is tightly regulated by the P-factor receptor (the GPCR) to which the P-factor pheromone binds. Only one copy of the sxa2 promoter exists in wild-type cells. Accordingly, it is possible to remove the naturally occurring sxa2 promoter and its associated sxa2 gene and replace it with a construct containing the reporter gene under the transcriptional control of the sxa2 promoter. This promoter-reporter construct may be integrated into the chromosome of the yeast cell.

[0038] Integrating the promoter-reporter gene construct into the chromosome of the yeast cell is advantageous because a known number of reporter genes are then found within each cell. If the promoter-reporter gene construct is placed on a plasmid, then the number of reporter genes in each cell may vary since the copy number of the plasmid may vary considerably and is not constant.

[0039] Inactivating the endogenous sxa2 gene, for example by at least partially deleting the sxa2 gene, can improve the sensitivity of the assay when P-factor is used to stimulate the GPCR. This is because inactivation of the carboxypeptidase reduces inactivation of the P-factor which may be used to stimulate the GPCR The reporter gene may be linked to any remaining sxa2 gene, for example to form a fusion protein. Alternatively, the entire sxa2 gene may be deleted and the reporter gene inserted in its place.

[0040] Preferably, the yeast cell used exhibits a stable mating type. Mating type in Sz. pombe is determined by information carried at the mat1 locus. Haploid cells containing the mat1-P segment, which contains the mat1-Pc and mat1-Pm genes, are ‘+’ (P or plus), and those with mat1-M, encoding mat1-Mc and mat1-Mm are ‘−’ (M or minus). Expression of mat1-Pc and mat1-Mc are required for expressing the genes that encode the pheromones and their receptors and hence establish the pheromone communication system. AU 4 mat1 genes are required for meiosis. There are two further mating loci, mat2 and mat3 where P and M information is stored but not expressed. In wild-type homothallic strains the information at mat2 and mat3 is frequently transferred to the mat1 locus and cells switch mating type approximately once every three generations. Cultures of such strains are therefore normally a mixture of both mating types (P and M). Even normal heterothallic strains are relatively unstable. Strains with a stable mating type can be generated by either deleting the mat2 and mat3 loci or by mutating the switching machinery, to produce a yeast cell exhibiting a stable mating phenotype (Davey, 1998).

[0041] Continued exposure to stimulus will lead to desensitisation of the signalling pathway. Several mechanisms are known to contribute to the desensitisation process. Selected mutations in the genes encoding proteins involved in desensitisation can lead to hypersensitivity and an inability to adapt to stimulation. This could be an advantage when using the strains in high throughput screens.

[0042] The yeast cell may be rgs1 deficient. Strains lacking rgs1 or having reduced Rgs1 (the product of the rgs1 gene) activity are hypersensitive to pheromone stimulation (Watson, et al., 1999).

[0043] The yeast cell may also be pmp1 deficient. The pmp1 gene encodes a dual specificity phosphatase that dephosphorylates the MAPK. Strains lacking this phosphatase exhibit an increased response following stimulation of the cells with a ligand for the GPCR.

[0044] The GPCR may be a naturally occurring yeast pheromone receptor. Alternatively, the receptor may be replaced, or contain in addition thereto, an heterologous receptor from another cell. When the GPCR is heterologous, it may be from any species other than Sz. pombe. The GPCR may be from a plant species or an animal species, particularly mammals, including economically significant non-human mammals. In one of the most important aspects of the invention, however, it will be a human GPCR. The GPCR may be any GPCR which it is desired to investigate by means of the invention. For example, the yeast cell may express an orphan receptor. That is, a receptor of unknown specific activity, but which has been identified by its homology to other GPCR receptors. The yeast cell may be modified to produce such orphan receptors using techniques known in the art. For example, a plasmid containing a nucleic acid sequence encoding for the orphan receptor operably linked to suitable promoter and regulatory sequences may be inserted into the yeast cell. The receptors may be modified to include a signal sequence that functions in Sz. pombe. Suitable signal sequences include those of Mam2, Map3 and of other gene products secreted by the Sz. pombe cells. If the wild-type heterologous GPCR cannot be made functional in Sz. pombe, it may be mutated for this purpose. In addition, the Sz. pombe cells may express endogenous GPCRs in a functional form.

[0045] The Sz. pombe cell must contain a G protein that is activated by the GPCR and can interact with the rest of the yeast intracellular signalling machinery. The endogenous Sz. pombe Gα subunit (Gpa1) may be able to couple the heterologous receptor to the intracellular signalling machinery. However, it may be necessary to engineer the Sz. pombe cell to produce a heterologous or chimeric G protein subunit (or subunits). At least 16 Gα subunits have been identified in mammals and a given GPCR usually activates only one or a small subset of Gα subunits. The amino- and carboxy-termini of Gα subunits do not share significant homology, but there are several generalisations that can be made. For example, the amino-termini have been implicated in association with Gβγ subunits and with membranes through N-terminal myristoylation. Interaction with the receptor is thought to involve the carboxy-termini as mutants lacking the 5 C-terminal residues of the Gα subunit fail to couple to their receptors (see, for example, Hirsch et al., 1991) and peptides based on the C-terminal region of the Gα subunit bind to receptors (Hamm et al., 1988; Palm et al., 1990; Rasenick et al., 1994). Work with chimeric Gα subunits further supports a critical role for the C-terminal residues in conferring receptor specificity (Voyno-Yasenetskaya et al., 1994; Liu et al., 1995). Thus, the A1 adenosine receptor naturally couples through Gi but can couple via a Gα chimera in which the C-terminal 4 residues of Gq were exchanged for those of Gi2 (Conklin et al., 1993) and the SST3 somatostatin receptor does not couple through Gs but can be coupled to adenylate cyclase by replacing the last S residues of Gs with those from Gi2 (Komatsuzaki et al., 1997).

[0046] Several reports have demonstrated that heterologously expressed GPCRs can couple to the intracellular signalling machinery in S. cerevisiae. Some of these receptors can interact with the endogenous Gα subunit (encoded by the GPA1 gene), including those for rat somatostatin (Price et al., 1995), rat A_(2A) adenosine (Price et al., 1996), human lysophosphatidic acid (Erickson et al., 1998) and human UDP-glucose (Chambers et al., 2000). Several other receptors, including that for human growth hormone releasing hormone, do not couple to the S. cerevisiae Gpa1 (Kajkowski et al., 1997). In order to attain coupling of these receptors to the intracellular signalling machinery, the S. cerevisiae Gα subunit can be replaced by a mammalian Gα subunit or by a chimeric Gα subunit in which the C-terminal region of the yeast Gα subunit is replaced with the equivalent region of the mammalian Gα subunit. Many examples of the use of chimeric Gα subunits are available (Price et al., 1995; Bass et al., 1996; Kajkowski et al., 1997; Klein et al., 1998; Baranski et al., 1999; Swift et al., 2000). In some instances, production of the chimeric Gα may involve the replacement of as few as S residues from thy C-terminus of the endogenous yeast Gα subunit with the equivalent residues from the mammalian Gα subunit. Such constructs are sometimes referred to as ‘Gα-transplants’. There are several reports describing the use of Gα-transplants in S. cerevisiae (Olesnicky et al., 1999; Brown et al., 2000; Chambers et al., 2000; Erlenbach et al., 2001).

[0047] The use of Gα-transplants based on the endogenous Sz. pombe Gα subunit may be used to improve the coupling of heterologous GPCRs. To ensure the correct stoichiometric relationship between the Gα-transplant and the Gβγ subunits, it may be necessary to replace the chromosomal copy of the natural Sz. pombe Gα gene (gpa1) with the equivalent construct encoding the Gα-transplant. However, it is also likely that expression from other promoters is compatible with coupling of the Gα subunits to the receptors.

[0048] Yeast cells of the invention containing the Gα-transplants, and vectors, such as plasmids, cosmids, etc. containing nucleic acid encoding the transplants are included in the invention.

[0049] The yeast cell may additionally comprise one or more nucleic acid molecules, such as plasmids, encoding for one or more peptides or proteins, to allow the peptide or protein to be assayed for its effect on GPCR-regulated activity of the reporter system Alternatively, one or more other chemical compounds may be added to determine the effect of the compound on reporter system activity.

[0050] Preferably, the yeast cells contain an auxotrophic marker that allows the selection of plasmids in the yeast cells. The leu1 mutation provides one such marker and makes growth of the cells dependent upon the addition of leucine or on the introduction of a plasmid containing the leu1 gene. Similar mutations can also be made to genes involved in the biogenesis of other nutrients (including histidine, lysine and arginine). Such markers include ade1, ade6, arg3, CAN1, his3, his7 and ura4, all of which are known in the art.

[0051] Plasmids containing the nucleic acid encoding for a peptide or protein to be assayed may contain one or more promoter, termination and processing signal sequences. Suitable promoters include the thiamine repressed nmt1 promoter. This is repressed by the presence of thiamine. Other suitable promoters include adh1 and fbp1, which are known in the art

[0052] The plasmid may also contain a yeast autonomous replication sequence (ARS) to enable the plasmid to replicate in the Sz. pombe cells.

[0053] A bacterial origin of replication (ori), together with one or more bacterial selection markers, such as the ampicillin or tetracycline-resistant genes, may also be included to allow the plasmid to be replicated within bacterial systems prior to insertion into yeast cells. Additionally, the plasmids may include one or more restriction endonuclease sites to enable nucleic acid sequences encoding the peptide or proteins of interest to be inserted. Most preferably, the nucleic acid sequence encoding the peptides or proteins is random and/or may be in the form of a conformational library. Such libraries are known in the art. This allows the production of random peptides to identify peptide regulators of interest. This also allows a library of yeast cells containing different peptides to be produced.

[0054] One or more nucleic acid sequences encoding for known peptides or proteins may be introduced into the cell. This allows, for example, a mammalian GPCR-regulated pathway to be reconstituted within a yeast cell.

[0055] The strain may additionally contain an ade6 mutation that helps to make diploid strains of Sz. pombe more stable. This is useful where diploid strains of yeast are desirable.

[0056] It is not intended that the modifications to Sz. pombe described above necessarily be the only modifications made to the cell. Further modifications can be made as required for tailoring the system to particular circumstances.

[0057] A further aspect of the invention provides an isolated nucleic acid molecule comprising a promoter regulatable by G-Protein Coupled Receptor (GPCR)-regulated signalling pathway in Schizosaccharomyces pombe, operatively linked to a reporter gene. It is preferred that the promoter be an sxa2 promoter or homologue or analogue thereof operatively linked to a reporter gene. The sxa2 promoter and/or reporter genes may be as previously described.

[0058] For the avoidance of doubt, in this context, by reporter gene we mean any detectable gene which is not a naturally occurring sxa2 gene.

[0059] A further aspect of the invention provides the use of a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising:

[0060] (i) a G-Protein Coupled Receptor (GPCR)-regulated signaling pathway which is derepressed during the mitotic phase of cell growth;

[0061] (ii) a reporter system for reporting a signal mediated by the GPCR-regulated signalling pathway; or

[0062] (iii) an isolated nucleic acid molecule as defined above to study GPCR-regulated activity.

[0063] A further aspect of the invention provides an assay comprising the use of a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising:

[0064] (i) a G-Protein Coupled Receptor (GPCR)-regulated signalling pathway which is derepressed during the mitotic phase of cell growth;

[0065] (ii) a reporter system for reporting a signal mediated by the GPCR-regulated signalling pathway; or

[0066] (iii) isolated DNA molecule as defined above.

[0067] The invention also provides a method of determining the effect of a compound on GPCR-regulated activity comprised in the steps of:

[0068] (i) providing a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising:

[0069] (a) a G-Protein Coupled Receptor (GPCR)-regulated signalling pathway which is derepressed during the mitotic phase of cell growth;

[0070] (b) a reporter system for reporting a signal mediated by the GPCR-regulated signalling pathway;

[0071] (ii) introducing the compound to the yeast cell; and

[0072] (iii) noting the output of the reporter system, for example by determining an amount of reporter gene product produced by the yeast cell.

[0073] The amount of reporter gene product or other reporter system output may be compared with a control yeast without the compound.

[0074] The invention also relates to the use of a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising:

[0075] (a) a G-Protein Coupled Receptor (GPCR)-regulated signalling pathway which is derepressed during the mitotic phase of cell growth;

[0076] (b) a reporter system for reporting a signal mediated by the GPCR-regulated signalling pathway;

[0077] to identify a compound which acts as the receptor. The compound may be the or a natural ligand for the receptor or be an agonist or antagonist (or partial agonist or partial antagonist). Such compounds affect the ability of the receptor to regulate the GPCR-regulated signalling pathway. The invention therefore encompasses the use of such a yeast cell containing an orphan GPCR to identify compounds that affect the ability of the orphan receptor to regulate the promoter is also provided.

[0078] The yeast cell, as defined above, may be used to identify a regulator or a mutant of a GPCR-regulated pathway.

[0079] The invention also provides a method of identifying a reagent that modulates GPCR-regulated signalling, comprising:

[0080] (i) providing a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising:

[0081] (a) a G-Protein Coupled Receptor (GPCR)-regulated signalling pathway which is derepressed during the mitotic phase of cell growth;

[0082] (b) a reporter system for reporting a signal mediated by the GPCR-regulated signalling pathway;

[0083] (ii) producing a random peptide within the yeast cell; and

[0084] (iii) noting the output of the reporter system, for example by measuring an amount of reporter gene product produced by the yeast cell.

[0085] A still further aspect of the invention provides a compound capable of modulating GPCR-regulated activity identified by a method according to the invention. Assay kits comprising a yeast cell or isolated nucleic acid molecule as defined above are also provided.

[0086] M-cells do not normally express the P-factor mating pheromone (encoded by the map2 gene). P-factor is an unmodified peptide of 23 amino acids that is initially synthesised as a precursor containing an N-terminal signal sequence and four tandem copies of the mature pheromone. The signal sequence is lost after targeting the precursor into the secretory pathway and the precursor is then processed into the individual subunits before being released into the medium. A plasmid-based map2 construct that contains a single copy of the pheromone peptide and is expressed under the control of the nmt1 promoter has been prepared. Reporter strains containing the plasmid secrete P-factor when grown in thiamine-free medium and this elicits an autocrine response in the yeast cell in which the P-factor produced by the cell stimulates the pheromone receptor expressed in the same cell.

[0087] A further aspect of the invention therefore provides a yeast cell containing such a construct. Restriction sites may be provided within the construct to allow the P-factor sequence to be replaced by an alternative peptide sequence that is then secreted into the medium. Introducing random sequences into this construct produces a library of yeast strains in which each individual releases a different peptide, and allows random peptides to be assayed for their ability to act as autocrine inducers.

[0088] Strains of cells of, and useful in, the invention may be termed “reporter strains”.

[0089] Another feature of the invention is that it provides a method of determining whether a GPCR is coupled to the intracellular signalling machinery even in the absence of a ligand. Such a method is particularly useful for investigating orphan GPCRs, for which the natural ligand may not be known. The method is based on the as yet unexplained observation that the ligand-independent reporter system response is higher in a cell lacking a coupled receptor than it is in a comparable cell having a coupled receptor.

[0090] According to this aspect of the invention, there is therefore provided a method of determining whether a G-Protein Coupled Receptor (GPCR) is coupled to a cell signalling pathway, the method comprising comparing the ligand-independent reporter system output of a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising:

[0091] (i) a G-Protein Coupled Receptor (GPCR)-regulated signalling pathway which is derepressed during the mitotic phase of cell growth;

[0092] (ii) a reporter system for reporting a signal mediated by the GPCR-regulated signalling pathway;

[0093] with the reporter system output of a reference cell which lacks a functional GPCR.

[0094] The reference cell, which itself forms another aspect of the invention, will generally be a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising:

[0095] (i) a G-Protein Coupled Receptor (GPCR)-regulated signalling pathway which is derepressed during the mitotic phase of cell growth, wherein the GPCR is absent or otherwise rendered non-functional;

[0096] (ii) a reporter system for reporting a signal mediated by the GPCR-regulated signalling pathway.

[0097] The reporter system will be expected to give an output indicative of higher activity from the reference cell than from the cell under investigation if the GPCR in the cell under investigation is coupled to the signalling pathway.

[0098] Preferred features of each aspect of the invention are as for each other aspect, mutatis mutandis.

[0099] The invention will now be described by way of example only, with reference to the following figures:

[0100]FIG. 1. Schematic diagram showing the identification and step-wise replacement of the sxa2 gene with ura4⁺, and the sxa2>ura4 and sxa2>lacZ reporter genes.

[0101]FIG. 2. Southern Blot of a PvuII and HindIII digest of the constructs shown schematically in FIG. 1.

[0102]FIG. 3. Schematic diagram of the arrangement of the map2 gene product

[0103]FIG. 4. Amino acid sequence of the map2 gene product.

[0104]FIG. 5. Schematic diagram showing the construction of a construct containing only one copy of the P-factor gene (the mono P construct).

[0105]FIG. 6. Amino acid sequence of the mono P construct.

[0106]FIG. 7A. Schematic diagram showing the replacement of a P-factor gene with a nucleic acid sequence encoding a random peptide, Where “n” is an unknown amino acid.

[0107]FIG. 7B. Amino acid sequence of the modified P-factor gene product encoding a random peptide, where “n” is an unknown amino acid.

[0108]FIG. 8. Positive and negative selection using the ura4 reporter gene: a) Growth of yeast cells on plates without uracil upon stimulation with P-factor; b) Inhibition of growth on FOA plates: Yeast cells are stimulated with 1, 10 and 100 units of P-factor.

[0109]FIG. 9. Growth of sxa2>ura4. yeast cells on plates without uracil. The yeast cells are stimulated with between 0.1 and 1000 units/ml. P-factor.

[0110]FIG. 10. Identification of mutants having enhanced sensitivity to P-factor stimulation. sxa2>ura4 cells were grown on plates lacking uracil.

[0111]FIG. 11. Identification and characterisation of rgs1 mutants using the sxa2>ura4 strain.

[0112]FIG. 12. Thiamine-inducible expression of P-factor using the sxa2>ura4 reporter strain and a thiamine-inducible P-factor construct.

[0113]FIG. 13. P-factor stimulation of β-galactosidase in the sxa2>lacZ reporter strain

[0114]FIG. 14. Coupling of the human CRH receptor in Sz. pombe strains containing various Gα-transplants.

[0115]FIG. 15. Demonstrating the coupling of a receptor in the absence of its ligand.

METHODS

[0116] All manipulations were by standard methods (see, for a general review, Davey et al., 1995). Reagents were obtained from common laboratory suppliers and used according to the manufacturer's recommendations. Unless stated otherwise, the polymerase chain reaction (PCR) was performed using Pwo DNA polymerase (from Pyrococcus woesei; supplied, by Boehringer Mannheim) as this has a 3′-5′ exonuclease (proof-reading) activity and reduced the introduction of errors during amplification. TAQ polymerase (from Thermus aquaticus; supplied by Boehringer Mannheim) was used for PCR with primers containing random sequences.

[0117] The yeast strains identified below are merely examples. Other suitable strains can be readily identified or produced using techniques known in the art.

[0118] Yeast Strains

[0119] JY271 is h⁻, cyrl::ura4, ade6-M216, leul-32, ura4-D18 and is equivalent to JZ300 (Maeda et al., 1990). This is an Mell but not stable and can switch mating type. The cyr1 gene (encoding adenylate cyclase) was disrupted by insertion of the ura4 gene (pDAC5), resulting in a cell which requires adenine and leucine for growth.

[0120] JY330 is mat1-P, Δmat2/3::LEU2⁻, leu1-32. The mat2-P and mat3-M donor mating cassettes were deleted by insertion of LEU2 (Klar and Miglio, 1986) and a LEU2⁻ isolate was then identified (Klar and Bonaduce, 1991).

[0121] JY444 is mat1-M, Δmat2/3::LEU2⁻, leu1-32, ura4-D18 and is a stable M-cell that requires leucine and uracil for growth.

[0122] The ura4 cassette used to disrupt the cyrl gene in JY271 was removed by standard techniques to create JY271B. JY271B is h⁻, cyrl-D51, ade6-M216, leul-32, ura4-D18.

[0123] This is an M-cell but not stable and can switch mating type. The cyr1 gene (adenylate cyclase) is disrupted. The cell requires adenine, leucine and uracil for growth.

[0124] JY271B was crossed with JY330 to generate JY361. JY361 is mat1-P, Δmat2/3::LEU2⁻, leu1-32, ade6-M216, ura4-D18, cyr1-D51. This is a stable P-cell which the cyr1 gene (adenylate cyclase) is disrupted. The cell requires adenine, leucine and uracil for growth.

[0125] JY361 was crossed with Y444 to generate JY486. JY486 is matl-M, Δmat2/3::LEU2⁻, leul-32, ade6-M216, ura4-D18, cyrl-D51. This is a stable M-cell in which the cyr1 gene (adenylate cyclase) is disrupted. The strain requires adenine, leucine and uracil for growth.

[0126] The sxa2 gene in JY486 was disrupted using a ura4⁺ cassette to generate JY522. The manipulation of the sxa2 gene is described in more detail below. The disruption cassette was a NcoI-to-BamHI fragment from JD883. JY522 is mat1-M, Δmat2/3::LEU2⁻, leu1-32, ade6-M216, ura4-D18, cyr1-D51, sxa2::ura4⁺. This is a stable M-cell in which the cyr1 gene (adenylate cyclase) is disrupted. The sxa2 gene (encodes a serine carboxypeptidase) is also disrupted. The strain requires adenine and leucine for growth.

[0127] The disrupted sxa2 gene in JY522 was replaced with the sxa2>lacZ reporter to generate JY546. The sxa2>lacZ reporter construct is from JD954. JY546 is matl-M, Δmat2/3::LEU2⁻, leul-32, ade6-M216, ura4-D18, cyrl-D51, sxa2>lacZ. This is a stable M-cell in which the cyrl gene (adenylate cyclase) is disrupted. The strain has an sxa2>lacZ reporter integrated at the sxa2 locus and expresses lacZ in response to pheromone stimulation. This strain requires adenine, leucine and uracil for growth.

[0128] The disrupted sxa2 gene in JY522 was also replaced with the sxa2>ura4 reporter to generate JY603. The sxa2>ura4 reporter construct is from JD929. JY603 is matl-M Δmat2/3::LEU2⁻, leul-32, ade6-M216, ura4-D18, cyrl-D51, sxa2>ura4, and is a stable M-cell. The cyr1 gene (adenylate cyclase) is disrupted. This has an sxa2>ura4 reporter integrated at the sxa2 locus and expresses ura4 in response to pheromone stimulation. The strain requires adenine and leucine for growth.

[0129] Constructing the sxa2>Reporter Strains

[0130] FIGS. 1 and 2 summarise the methods used to manipulate the sxa2 gene and promoter.

[0131] The sxa2 ORF was first replaced with a 1.8 kb Sz. pombe ura4+ cassette (Grimm et al., 1988). The complete sxa2 locus was amplified by PCR using the sense primer JO760 (ggggggtacCATGGCTAGAAATCCGCCATTGTGTG; lower-case letters are not complementary to sxa2 but the oligonucleotide includes a KpnI site [ggtac*C] and an NcoI site [c*CATGG] where digestion leaves ends that are fully homologous to the. chromosomal sequence) and the antisense primer JO683 (CTTCTCGTAAAGGCACATTGACGG, complementary to a region immediately downstream of the BamHI site at position 2043). The resulting PCR product was cloned into the KpnI and BamHI sites of pSP72 (Promega) to generate JD808 (pSP72 containing the sxa2 locus SEQ ID 31). This was used as template for PCR with JO746 (TGAAAAGAGAGACAATG; antisense primer complementary to a region immediately upstream of the ATG initiator codon for sxa2) and JO745 (TAAAAGTTTAATATC; sense primer complementary to a region that includes the TAA stop codon for sxa2) and the product ligated to the ura4⁺ cassette (to generate JD857, pSP72 containing a construct suitable for disruption of sxa2 SEQ ID 32) or to PCR products corresponding to either the lacZ ORF (to generate JD954, pSP72 containing the sxa2>lacZ reporter construct SEQ ID 33) or the ura4 ORF (to generate JD929, pSP72 containing the sxa2>ura4 reporter construct SEQ ID 34). The lacZ ORF was prepared by amplification using the sense primer JO660 (ATGCAGCTGGCACGACAGGTTTCCCGAC; includes the ATG initiator codon and next 25 bases of the lacZ ORF) and the antisense primer JO661 (TTTTTGACACCAGACCAACTGGTAATGGTAGC; complementary to the 3′ end of the lacZ ORF but lacks the stop anticodon). The ura4 ORF was prepared by amplification using the sense primer JO828 (ATGGATGCTAGAGTATTTC; includes the ATG initiator codon and next 16 bases of the ura4 ORF) and the antisense primer JO759 (ATGCTGAGAAAGTCTTTGC; complementary to the 3′ end of the ura4 ORF but lacks the stop anticodon).

[0132] JY486 (a mating stable M-cell lacking cyrl) was transformed with a NcoI-BamHI fragment corresponding to the sxa2::ura4⁺ construct (isolated from JD857), and stable Ura4⁺ transformants were initially screened by PCR and replacement of the sxa2 locus was confirmed by Southern blot (FIG. 2). A correct sxa2::ura4⁺ disruptant (JY522) was then transformed with the NcoI-BamHI fragments corresponding to the sxa2>lacZ reporter (isolated from JD954) or the sxa2>ura4 reporter (isolated from JD929). Stable Ura⁻ transformants were selected by their ability to grow in the presence of 5′fluoro-orotic acid (Boeke et al., 1987) and homologous integration of the reporter constructs at the sxa2 locus was confirmed by Southern blot for JY546 (sxa2>lacZ) and JY603 (sxa2>ura4). Southern blot analysis was performed on genornic DNA digested with PvuII & HindIII and a probe corresponding to the 5′ untranslated region of sxa2.

[0133] Constructing the Gα-Transplants

[0134] This was undertaken using techniques well known in the art. A SpeI-PstI fragment from gpa1 (SEQ ID 1, 15) was cloned into the SpeI and PstI sites of the plasmid pKS-Bluescript (Stratagene). This 324 bp fragment contains the last 24 residues of Gpa1 and 250 base pairs from the 3′ untranslated region of the gpa1 gene. The resulting clone (JD1647) was then used as template for a series of polymerase chain reactions using oligonucleotide primers that made the desired changes to the residues at the C-terminus of Gpa1. Each reaction used the antisense primer JO1354 (TAGATTGTTGGACATAATCGTATCTTGAACGG; complementary to a region from position 1206 to position 1175 relative to the intiator ATG of gpa1) and an appropriate sense primer that introduced the desired changes and was complementary to the region immediately downstream of the Gpa1 open reading frame; JO1344 for the Gαq-transplant (gaatataatcttgttTAGATGAATTMTCCTTAAC, lower case letters, change the last 5 residues of Sz. pombe Gpa1 to EYNLV), JO1345 for the Gαs-transplant (caatatgaacttcttTAGATGAATTTTTCCTTAAC; change last 5 residues of Gpa1 to QYELL), JO1346 for the Gαo-transplant (ggatgcggactttatTAGATGAATTTTTCCTTAAC, change last 5 residues of Gpa1 to GCGLY), JO1347 for the Gαi2-transplant (gattgcggactttttTAGATGAATTTlTCCTTAAC, change last 5 residues of Gpa1 to DCGLF), JO1348 for the Gαi3-transplant (gaatgcggactttatTAGATGAATTTTTTCCAAC, change last 5 residues of Gpa1 to ECGLY), JO1349 for the Gαz-transplant (tatattggactttgcTAGATGAATTTTTCCTTAAC, change last 5 residues of Gpa1 to YIGLC), JO1350 for the Gα12-transplant (gatattatgcttcaaTAGATGAATTTTTCCTTAAC, change last 5 residues of Gpa1 to DIMLQ), JO01351 for the Gα13-transplant (caacttatgcttcaaTAGATGAATTTTTCCTTAAC, change last 5 residues of Gpa1 to QLMLQ), JO1352 for the Gα14-transplant (gaatttaatcttgttTAGATGAATTTTCCTTAAC, lower case letters change the last 5 residues of Gpa1 to EFNLV) and JO1353 for the Gα16-transplant (gaaattaatcttcttTAGATGAATTTTTCCTTAAC, change last 5 residues Gpa1 to EINLL).

[0135] The PCR products were sequenced to confirm that the correct changes had been made and were then used to replace the equivalent SpeI-PstI fragment from JD1645 (pSP71-Gpa1). JD1645 contains the complete gpa1 sequence from an EcoRI site at position −676 (relative to the initiator ATG) to a BglII site at position 1938. This generated a series of plasmids containing the modified gpa1 sequences; JD1649 (Gαq-transplant SEQ ID 17, 03), JD1650 (Gαs-transplant SEQ ID 16, 02), JD1651 (Gαo-transplant SEQ ID 18, 04), JD1652 (Gαi2-transplant SEQ ID 19, 05), JD1653 (Gαi3-transplant SEQ ID 20, 06), JD1654 (Gαz-transplant SEQ ID 21, 07), JD1655 (Gα12-transplant SEQ ID 22, 08), JD1656 (Gα13-transplant SEQ ID 23, 09), JD1657 (Gα14-transplant SEQ ID 24, 10) and JD1658 (Gα16-transplant SEQ ID 25, 11). The coding regions for the different Gα-transplants were isolated as EcoRI-BglII fragments and used separately to transform the yeast strain JY1170. JY1170 is matl-M, Δmat2/3::LEU2⁻, leul-32, ade6-M216, ura4-D18, cyrl-D51, mam2-D10, gpa1::ura4⁺, sxa2>lacZ. This is a derivative of the standard JY546 reporter strain but it lacks the mam2 gene (encodes the P-factor receptor) and the gpa1 gene has been disrupted by insertion of a ura4⁺ cassette. Ura⁻ transformants were selected on fluoro-orotic acid and Southern blot analyses were used to confirm integration of the Gα-transplant constructs at the gpa1 locus. This generated a series of Sz. pombe sxa2>lacZ reporter strains lacking the mam2 pheromone receptor but containing integrated Gα-transplants; JY1165 (Gαq-transplant), JY1157 (Gαs-transplant), JY1158 (Gαo-transplant), JY1159 (Gαi2-transplant), JY1160 (Gαi3-transplant), JY1161 (Gαz-transplant), JY1162 (Gα12-transplant), JY1163 (Gα13-transplant), JY1164 (Gα14-transplant) and JY1167 (Gα16-transplant).

[0136] Generating Peptides for Autocrine Signalling

[0137] M-cells do not normally express the P-factor mating pheromone (encoded by the map2 gene). P-factor is an unmodified peptide of 23 amino acids that is initially synthesised as a precursor containing an N-terminal signal sequence and four tandem copies of the mature pheromone. The signal sequence is lost after targeting the precursor into the secretory pathway and the precursor is then processed into the individual subunits before being released into the medium. A plasmid-based map2 construct that contains a single copy of the pheromone peptide and is expressed under the control of the thiamine-regulated nmtl promoter shown schematically in FIGS. 3 to 6 was prepared.

[0138] This was undertaken using techniques well known in the art. Reporter strains containing the plasmid secrete P-factor when grown in thiamine-free medium (the nmtl promoter is on) and this elicits an autocrine response in the strain. Restriction sites within the construct allow the P-factor sequence to be replaced by an alternative peptide sequence that would then be secreted into the medium (FIGS. 7A and 7B). Introducing random sequences into this construct produces a library of strains in which each individual releases a different peptide. This allows ligands capable of binding to the pheromone receptor or another GPCR to be identified.

[0139] Expression and Application of Reporter Gene Constructs

[0140] Demonstration of a sxa2>ura4 Reporter Construct

[0141] JY603 yeast cells containing the construct were spread as a confluent layer of cells (about 10⁷ cells on each plate) on DMM medium lacking uracil. Paper disks were placed on the dried surface of the cells and aliquots containing different amounts of P-factor were added to each disk. The plates were then incubated at 29° C. for 3 days. FIG. 8A shows that cells are not normally able to grow in the absence of uracil but the P-factor induces expression of the sxa2>ura4 reporter and allows a growth of cells around the disks. The halo is largest around the disk containing 100 units of P-factor.

[0142] This is also demonstrated in FIG. 4, except that a series of plates containing different concentrations of P-factor were used. AU of the plates received the same number of yeast cells (about 2,000 cells per plate). The cells are not normally able to grow in the absence of uracil but the P-factor induces expression of the sxa2>ura4 reporter and allows cells to form colonies. There are no colonies on the plates containing 0.1 or 1.0 units/ml. but colonies form on plates containing P-factor with at least 10 units/ml.

[0143]FIG. 8B shows plates which contain uracil and 5-fluoro-orotic acid (FOA). Cells not expressing ura4 are able to grow on these plates but those expressing ura4 convert the FOA into a toxic compound and die. There are clear halos of no growth around the disk containing the P-factor. The halo is largest around the disk containing 100 units of P-factor.

[0144] Identification of Mutants

[0145] The sxa2>ura4 reporter system allows the identification of mutants. Cells can be randomly mutagenised and spread on plates containing P-factor at 0.1 units/ml to identify mutations that make the cells more sensitive to stimulation. FIG. 10 shows two of these mutations. This approach can also be used to identify mutant forms of various proteins involved in regulating the signalling pathway as shown in FIG. 11.

[0146] The sxa2>ura4 reporting strain was randomly mutagenised and then spread on plates lacking uracil but containing P-factor at 0.1 units/ml. The wild-type cells do not normally grow on these plates, since they require P-factor at a concentration of at least 10 units/ml. A number of mutants that had increased sensitivity to signalling and were now able to grow at a low level of P-factor were identified. Two of these mutants have been characterised as being rgs1 and pmp1. The pmp1 mutant does not grow on plates lacking P-factor but grows on a very low level of P-factor. This demonstrates that it is a hypersensitive mutant. In contrast, the rgs1 mutant grows even in the absence of P-factor, showing that it is a constitutive responder (that is, it expresses the reporter gene in the absence of stimulation by ligand).

[0147] The Applicants mutated the cloned rgs1 gene to isolate mutant forms of the protein with altered properties and screened for isolates that were either gain of function mutants (have increased activity relative to the normal Rgs1 protein) or dominant negative mutants (inactive mutants that inhibit the activity of the normal Rgs1 protein in the same cell).

[0148] Autocrine Signalling

[0149] The inventors modified a version of the map2 gene that encodes the P-factor precursor was modified so that it contained a single copy of the P-factor (“mono P”). This is cloned into a plasmid so that expression of the P-factor was under the control of the thiamine-repressible nmtl promoter. The plasmid was introduced into M-cells which do not normally produce P-factor but are able to respond to P-factor. The cells were spread on plates lacking uracil but containing either no thiamine or 5 μM thiamine (see FIG. 12). The thiamine induces expression and release of the P-factor, causing autocrine signalling of the cell. Thins results in the expression of the sxa2>ura4 reporter.

[0150] The lacZ Reporter

[0151] The sxa2>lacZ reporter strain was grown in the presence of varying amounts of P-factor. The amount of β-galactosidase released was assayed using o-nitrophenyl-β-D-galactopyranoside and measuring the amount of product at OD₄₂₀. FIG. 13 shows the effect of adding P-factor over time and with increasing concentration The concentration-dependent assay was measured 16 hours after adding the pheromone.

[0152] Coupling of a Human GPCR

[0153] By way of example, the human receptor for corticotrophin releasing hormone (CRH, also known as corticotrophin releasing factor or CRF) was expressed in the Sz. pombe sxa2>lacZ reporter strains containing either Gpa1 or the various Gα-transplants. The yeast strains were transformed with pREP3X:CRH-R1 (SEQ ID 30), a plasmid that places the CRH receptor (see SEQ ID 28 and SEQ ID 14) type 1α under the control of the nmt1 promoter. Transformants were grown in the absence of thiamine (to allow expression of the receptor) and then exposed to CRH at 10⁻⁶ M (control cells were exposed to solvent lacking CRH). The amount of β-galactosidase released was assayed after 16 hours using o-nitrophenyl-β-D-galactopyranoside (FIG. 14). A low level of coupling was observed with the endogenous Gpa1 but this was considerably improved in the Gαs- and Gα16-transplants.

[0154] CRH is a 41-residue peptide that is a major regulator of the body’s stress axis. Although it has several functions, its best characterised role is in initiating pituitary-adrenal responses to stress, an effect mediated through CRH-R1α (Vale et al., 1981). This receptor normally functions through Gαs, resulting in activation of adenylate 2cyclase and increased levels of cAMP (Giguere et al., 1982; Bilezikjan and Vale, 1983; Grammatopoulos et al., 1996). The observed coupling to the Gαs-transplant is consistent with the activity of the CRH-R1α receptor in mammalian cells. Gα16 is known to interact with a wide range of GPCRs (Milligan et al., 1996).

[0155] There are no reports of the coupling of the human CRH receptor to the signalling machinery in the budding yeast S. cerevisiae and a direct comparison with the Sz. pombe reporter strains reported here is therefore not possible. It is perhaps significant however that a peptide ligand similar to CRH appears unable to gain access to receptors at the surface of S. cerevisiae cells (Baranski et al., 1999). The C5a chemoattractant receptor is functional in S. cerevisiae but can only be stimulated by its ligand (a 74-residue peptide) when both the receptor and the ligand are expressed in the same cell. Such autocrine stimulation is required because the C5a ligand is unable to traverse the S. cerevisiae cell wall.

[0156]Sz. pombe is also surrounded by a cell wall but it has a very different structure to that surrounding S. cerevisiae (for reviews, see, Osurmi, 1998; Smits et al., 1999) and previous studies of intoxication by diphtheria toxin demonstrated that the two have quite different permeability properties. Diphtheria toxin, secreted by certain strains of Corynebacterium diphtheriae, catalyses the ADP-ribosylation of eukaryotic aminoacyl transferase II (EF-2) using NAD as substrate. This reaction forms the basis for its toxicity toward eukaryotic organisms. Intoxication requires the entry of the toxin into the cytoplasm after internalisation by endocytosis. Studies have investigated the effects of diphtheria toxin on protein synthesis in S. cerevisiae (Murakami et al., 1982) and Sz. pombe (Davey, 1991). Although the Sz. pombe cells were sensitive to the toxin, intact S. cerevisiae cells were resistant to its effects. In contrast, S. cerevisiae spheroplasts (cells in which the cell wall has been enzymatically removed) were sensitive to the toxin, suggesting that the failure of the toxin to enter intact cells was due to its inability to cross the cell wall. Diphtheria toxin is a heterodimer composed of an N-terminal A fragment (molecular weight 24,000 daltons) that is enzymatically active and a C-terminal B fragment (molecular weight 39,000 daltons) that has no apparent enzymatic activity but is required for toxicity.

[0157] The apparent greater permeability of the cell wall in Sz. pombe night be invaluable for the study of receptors with large or complex ligands and could provide an advantage over the use of S. cerevisiae in such situations.

[0158] Demonstrating Coupling of a Receptor in the Absence of its Ligand

[0159] The lack of available ligands for orphan GPCRs makes it difficult to confirm that such receptors are coupled to the signalling machinery. High throughput screens are therefore performed without the confidence that a ligand which would normally stimulate the receptor would be identified as being active. The inventors have observed an interesting feature of the Sz. pombe reporter strains that can indicate whether a GPCR is coupled to the intracellular signalling machinery even in the absence of its ligand. Such knowledge generates confidence prior to performing high throughput screens. An example of this feature is shown in FIG. 15. When a S. pombe sxa2>lacZ reporter strain expressing the normal P-factor pheromone receptor is exposed to P-factor, there is a ligand-dependent induction of β-galactosidase. As expected, a similar strain lacking the P-factor receptor fails to exhibit ligand-dependent induction of the sxa2>lacZ reporter. However, the ligand-independent expression of the sxa2>lacZ reporter (i.e. the level of β-galactosidase activity observed in the absence of P-factor) is considerably higher in the strain lacking the P-factor receptor than in the strain containing the P-factor receptor. Expressing the human CRH-R1α receptor in the strain lacking the P-factor receptor reduces the ligand-independent expression of the sxa2>lacZ reporter back to the levels observed in the strain containing the P-factor receptor. As there is no P-factor receptor in this strain, addition of P-factor fails to induce further expression of the sxa2>lacZ reporter.

[0160] This observation is not limited either to the particular reporter system or to the CRH-R1α receptor and has been observed with many other GPCRs that were then subsequently shown to be coupled to the Sz. pombe signalling machinery. A molecular explanation for this effect is not available but it could simply reflect the ability of the receptor to sequester the heterotrimeric G proteins and prevent inappropriate activation of the downstream effector protein(s). Whatever the explanation, the ability of a receptor to reduce the ligand-independent expression of β-galactosidase appears to reflect its ability to couple to the Sz. pombe signalling machinery.

[0161] Applications

[0162] Identify Agonists and Antagonists for GPCRs, Including Orphan GPCRs

[0163] Reporter strains expressing either a characterised or an orphan receptor can be used in a variety of assays to identify ligands that affect signalling through the receptors. Agonists will elicit a response in the strain while antagonists could be identified by their ability to inhibit stimulation by a ligand known to activate the receptor. Both peptides and small molecules can be screened and assays might be either liquid- or plate-based, depending on the reporter gene used. Screens for peptide ligands could exploit the autocrine signalling of strains producing a library of random peptides.

[0164] Identify Intracellular Signal Regulators and Modified Regulators with Altered Activities

[0165] Regulators of the intracellular response pathway can be identified by their ability to influence signalling in the reporter strains. Over-expression of these proteins will either reduce or increase signalling depending on whether they are positive or negative regulators. A number of mammalian regulators are known to be active in yeast. Regulators identified through these screens can then be mutagenised and the reporter strains used to identify isolates with altered activities. Gain-of-function mutants, for example, would have increased abilities to regulate signalling while dominant-negative mutants would not only be inactive but would also inhibit the activity of the wild type regulator. These mutants could then be introduced back into mammalian systems to assess their ability to regulate other signalling pathways.

[0166] Identify Reagents that Modulate Signalling

[0167] Random peptides can be expressed in the cytoplasm of the reporter strains and assayed for their ability to regulate signalling. These ‘peptamers’ could interact directly with components from the signalling pathway or might exert their effect through the intracellular signal regulators mentioned earlier. The screen is not limited to peptide regulators and would also identify small molecules that could influence signalling.

[0168]Schizosaccharomyces pombe strain JY546 was deposited under the Budapest Treaty at the National Collection of Yeast Cultures, Norwich,. United Kingdom on 27 Oct. 2000. It has been given Accession Number NCYC 2984.

[0169] References

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1 53 1 407 PRT Schizosaccharomyces pombe 1 Met Gly Cys Met Ser Ser Lys Tyr Ala Asp Thr Ser Gly Gly Glu Val 1 5 10 15 Ile Gln Lys Lys Leu Ser Asp Thr Gln Thr Ser Asn Ser Ser Thr Thr 20 25 30 Gly Ser Gln Asn Ala Arg Val Pro Val Leu Glu Asn Trp Leu Asn Ile 35 40 45 Val Leu Arg Gly Lys Pro Gln Asn Val Glu Ser Ser Gly Val Arg Val 50 55 60 Lys Gly Asn Ser Thr Ser Gly Gly Asn Asp Ile Lys Val Leu Leu Leu 65 70 75 80 Gly Ala Gly Asp Ser Gly Lys Thr Thr Ile Met Lys Gln Met Arg Leu 85 90 95 Leu Tyr Ser Pro Gly Phe Ser Gln Val Val Arg Lys Gln Tyr Arg Val 100 105 110 Met Ile Phe Glu Asn Ile Ile Ser Ser Leu Cys Leu Leu Leu Glu Ala 115 120 125 Met Asp Asn Ser Asn Val Ser Leu Leu Pro Glu Asn Glu Lys Tyr Arg 130 135 140 Ala Val Ile Leu Arg Lys His Thr Ser Gln Pro Asn Glu Pro Phe Ser 145 150 155 160 Pro Glu Ile Tyr Glu Ala Val His Ala Leu Thr Leu Asp Thr Lys Leu 165 170 175 Arg Thr Val Gln Ser Cys Gly Thr Asn Leu Ser Leu Leu Asp Asn Phe 180 185 190 Tyr Tyr Tyr Gln Asp His Ile Asp Arg Ile Phe Asp Pro Gln Tyr Ile 195 200 205 Pro Ser Asp Gln Asp Ile Leu His Cys Arg Ile Lys Thr Thr Gly Ile 210 215 220 Ser Glu Glu Thr Phe Leu Leu Asn Arg His His Tyr Arg Phe Phe Asp 225 230 235 240 Val Gly Gly Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu 245 250 255 Asn Val Thr Ala Leu Leu Phe Leu Val Ser Leu Ala Gly Tyr Asp Gln 260 265 270 Cys Leu Val Glu Asp Asn Ser Gly Asn Gln Met Gln Glu Ala Leu Leu 275 280 285 Leu Trp Asp Ser Ile Cys Asn Ser Ser Trp Phe Ser Glu Ser Ala Met 290 295 300 Ile Leu Phe Leu Asn Lys Leu Asp Leu Phe Lys Arg Lys Val His Ile 305 310 315 320 Ser Pro Ile Gln Lys His Phe Pro Asp Tyr Gln Glu Val Gly Ser Thr 325 330 335 Pro Thr Phe Val Gln Thr Gln Cys Pro Leu Ala Asp Asn Ala Val Arg 340 345 350 Ser Gly Met Tyr Tyr Phe Tyr Leu Lys Phe Glu Ser Leu Asn Arg Ile 355 360 365 Ala Ser Arg Ser Cys Tyr Cys His Phe Thr Thr Ala Thr Asp Thr Ser 370 375 380 Leu Leu Gln Arg Val Met Val Ser Val Gln Asp Thr Ile Met Ser Asn 385 390 395 400 Asn Leu Gln Ser Leu Met Phe 405 2 407 PRT Artificial Sequence Description of Artificial Sequence G alpha transplant 2 3 407 PRT Artificial Sequence Description of Artificial Sequence G alpha transplant 3 Met Gly Cys Met Ser Ser Lys Tyr Ala Asp Thr Ser Gly Gly Glu Val 1 5 10 15 Ile Gln Lys Lys Leu Ser Asp Thr Gln Thr Ser Asn Ser Ser Thr Thr 20 25 30 Gly Ser Gln Asn Ala Arg Val Pro Val Leu Glu Asn Trp Leu Asn Ile 35 40 45 Val Leu Arg Gly Lys Pro Gln Asn Val Glu Ser Ser Gly Val Arg Val 50 55 60 Lys Gly Asn Ser Thr Ser Gly Gly Asn Asp Ile Lys Val Leu Leu Leu 65 70 75 80 Gly Ala Gly Asp Ser Gly Lys Thr Thr Ile Met Lys Gln Met Arg Leu 85 90 95 Leu Tyr Ser Pro Gly Phe Ser Gln Val Val Arg Lys Gln Tyr Arg Val 100 105 110 Met Ile Phe Glu Asn Ile Ile Ser Ser Leu Cys Leu Leu Leu Glu Ala 115 120 125 Met Asp Asn Ser Asn Val Ser Leu Leu Pro Glu Asn Glu Lys Tyr Arg 130 135 140 Ala Val Ile Leu Arg Lys His Thr Ser Gln Pro Asn Glu Pro Phe Ser 145 150 155 160 Pro Glu Ile Tyr Glu Ala Val His Ala Leu Thr Leu Asp Thr Lys Leu 165 170 175 Arg Thr Val Gln Ser Cys Gly Thr Asn Leu Ser Leu Leu Asp Asn Phe 180 185 190 Tyr Tyr Tyr Gln Asp His Ile Asp Arg Ile Phe Asp Pro Gln Tyr Ile 195 200 205 Pro Ser Asp Gln Asp Ile Leu His Cys Arg Ile Lys Thr Thr Gly Ile 210 215 220 Ser Glu Glu Thr Phe Leu Leu Asn Arg His His Tyr Arg Phe Phe Asp 225 230 235 240 Val Gly Gly Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu 245 250 255 Asn Val Thr Ala Leu Leu Phe Leu Val Ser Leu Ala Gly Tyr Asp Gln 260 265 270 Cys Leu Val Glu Asp Asn Ser Gly Asn Gln Met Gln Glu Ala Leu Leu 275 280 285 Leu Trp Asp Ser Ile Cys Asn Ser Ser Trp Phe Ser Glu Ser Ala Met 290 295 300 Ile Leu Phe Leu Asn Lys Leu Asp Leu Phe Lys Arg Lys Val His Ile 305 310 315 320 Ser Pro Ile Gln Lys His Phe Pro Asp Tyr Gln Glu Val Gly Ser Thr 325 330 335 Pro Thr Phe Val Gln Thr Gln Cys Pro Leu Ala Asp Asn Ala Val Arg 340 345 350 Ser Gly Met Tyr Tyr Phe Tyr Leu Lys Phe Glu Ser Leu Asn Arg Ile 355 360 365 Ala Ser Arg Ser Cys Tyr Cys His Phe Thr Thr Ala Thr Asp Thr Ser 370 375 380 Leu Leu Gln Arg Val Met Val Ser Val Gln Asp Thr Ile Met Ser Asn 385 390 395 400 Asn Leu Glu Tyr Asn Leu Val 405 4 407 PRT Artificial Sequence Description of Artificial Sequence G alpha transplant 4 Met Gly Cys Met Ser Ser Lys Tyr Ala Asp Thr Ser Gly Gly Glu Val 1 5 10 15 Ile Gln Lys Lys Leu Ser Asp Thr Gln Thr Ser Asn Ser Ser Thr Thr 20 25 30 Gly Ser Gln Asn Ala Arg Val Pro Val Leu Glu Asn Trp Leu Asn Ile 35 40 45 Val Leu Arg Gly Lys Pro Gln Asn Val Glu Ser Ser Gly Val Arg Val 50 55 60 Lys Gly Asn Ser Thr Ser Gly Gly Asn Asp Ile Lys Val Leu Leu Leu 65 70 75 80 Gly Ala Gly Asp Ser Gly Lys Thr Thr Ile Met Lys Gln Met Arg Leu 85 90 95 Leu Tyr Ser Pro Gly Phe Ser Gln Val Val Arg Lys Gln Tyr Arg Val 100 105 110 Met Ile Phe Glu Asn Ile Ile Ser Ser Leu Cys Leu Leu Leu Glu Ala 115 120 125 Met Asp Asn Ser Asn Val Ser Leu Leu Pro Glu Asn Glu Lys Tyr Arg 130 135 140 Ala Val Ile Leu Arg Lys His Thr Ser Gln Pro Asn Glu Pro Phe Ser 145 150 155 160 Pro Glu Ile Tyr Glu Ala Val His Ala Leu Thr Leu Asp Thr Lys Leu 165 170 175 Arg Thr Val Gln Ser Cys Gly Thr Asn Leu Ser Leu Leu Asp Asn Phe 180 185 190 Tyr Tyr Tyr Gln Asp His Ile Asp Arg Ile Phe Asp Pro Gln Tyr Ile 195 200 205 Pro Ser Asp Gln Asp Ile Leu His Cys Arg Ile Lys Thr Thr Gly Ile 210 215 220 Ser Glu Glu Thr Phe Leu Leu Asn Arg His His Tyr Arg Phe Phe Asp 225 230 235 240 Val Gly Gly Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu 245 250 255 Asn Val Thr Ala Leu Leu Phe Leu Val Ser Leu Ala Gly Tyr Asp Gln 260 265 270 Cys Leu Val Glu Asp Asn Ser Gly Asn Gln Met Gln Glu Ala Leu Leu 275 280 285 Leu Trp Asp Ser Ile Cys Asn Ser Ser Trp Phe Ser Glu Ser Ala Met 290 295 300 Ile Leu Phe Leu Asn Lys Leu Asp Leu Phe Lys Arg Lys Val His Ile 305 310 315 320 Ser Pro Ile Gln Lys His Phe Pro Asp Tyr Gln Glu Val Gly Ser Thr 325 330 335 Pro Thr Phe Val Gln Thr Gln Cys Pro Leu Ala Asp Asn Ala Val Arg 340 345 350 Ser Gly Met Tyr Tyr Phe Tyr Leu Lys Phe Glu Ser Leu Asn Arg Ile 355 360 365 Ala Ser Arg Ser Cys Tyr Cys His Phe Thr Thr Ala Thr Asp Thr Ser 370 375 380 Leu Leu Gln Arg Val Met Val Ser Val Gln Asp Thr Ile Met Ser Asn 385 390 395 400 Asn Leu Gly Cys Gly Leu Tyr 405 5 407 PRT Artificial Sequence Description of Artificial Sequence G alpha transplant 5 Met Gly Cys Met Ser Ser Lys Tyr Ala Asp Thr Ser Gly Gly Glu Val 1 5 10 15 Ile Gln Lys Lys Leu Ser Asp Thr Gln Thr Ser Asn Ser Ser Thr Thr 20 25 30 Gly Ser Gln Asn Ala Arg Val Pro Val Leu Glu Asn Trp Leu Asn Ile 35 40 45 Val Leu Arg Gly Lys Pro Gln Asn Val Glu Ser Ser Gly Val Arg Val 50 55 60 Lys Gly Asn Ser Thr Ser Gly Gly Asn Asp Ile Lys Val Leu Leu Leu 65 70 75 80 Gly Ala Gly Asp Ser Gly Lys Thr Thr Ile Met Lys Gln Met Arg Leu 85 90 95 Leu Tyr Ser Pro Gly Phe Ser Gln Val Val Arg Lys Gln Tyr Arg Val 100 105 110 Met Ile Phe Glu Asn Ile Ile Ser Ser Leu Cys Leu Leu Leu Glu Ala 115 120 125 Met Asp Asn Ser Asn Val Ser Leu Leu Pro Glu Asn Glu Lys Tyr Arg 130 135 140 Ala Val Ile Leu Arg Lys His Thr Ser Gln Pro Asn Glu Pro Phe Ser 145 150 155 160 Pro Glu Ile Tyr Glu Ala Val His Ala Leu Thr Leu Asp Thr Lys Leu 165 170 175 Arg Thr Val Gln Ser Cys Gly Thr Asn Leu Ser Leu Leu Asp Asn Phe 180 185 190 Tyr Tyr Tyr Gln Asp His Ile Asp Arg Ile Phe Asp Pro Gln Tyr Ile 195 200 205 Pro Ser Asp Gln Asp Ile Leu His Cys Arg Ile Lys Thr Thr Gly Ile 210 215 220 Ser Glu Glu Thr Phe Leu Leu Asn Arg His His Tyr Arg Phe Phe Asp 225 230 235 240 Val Gly Gly Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu 245 250 255 Asn Val Thr Ala Leu Leu Phe Leu Val Ser Leu Ala Gly Tyr Asp Gln 260 265 270 Cys Leu Val Glu Asp Asn Ser Gly Asn Gln Met Gln Glu Ala Leu Leu 275 280 285 Leu Trp Asp Ser Ile Cys Asn Ser Ser Trp Phe Ser Glu Ser Ala Met 290 295 300 Ile Leu Phe Leu Asn Lys Leu Asp Leu Phe Lys Arg Lys Val His Ile 305 310 315 320 Ser Pro Ile Gln Lys His Phe Pro Asp Tyr Gln Glu Val Gly Ser Thr 325 330 335 Pro Thr Phe Val Gln Thr Gln Cys Pro Leu Ala Asp Asn Ala Val Arg 340 345 350 Ser Gly Met Tyr Tyr Phe Tyr Leu Lys Phe Glu Ser Leu Asn Arg Ile 355 360 365 Ala Ser Arg Ser Cys Tyr Cys His Phe Thr Thr Ala Thr Asp Thr Ser 370 375 380 Leu Leu Gln Arg Val Met Val Ser Val Gln Asp Thr Ile Met Ser Asn 385 390 395 400 Asn Leu Asp Cys Gly Leu Phe 405 6 407 PRT Artificial Sequence Description of Artificial Sequence G alpha transplant 6 Met Gly Cys Met Ser Ser Lys Tyr Ala Asp Thr Ser Gly Gly Glu Val 1 5 10 15 Ile Gln Lys Lys Leu Ser Asp Thr Gln Thr Ser Asn Ser Ser Thr Thr 20 25 30 Gly Ser Gln Asn Ala Arg Val Pro Val Leu Glu Asn Trp Leu Asn Ile 35 40 45 Val Leu Arg Gly Lys Pro Gln Asn Val Glu Ser Ser Gly Val Arg Val 50 55 60 Lys Gly Asn Ser Thr Ser Gly Gly Asn Asp Ile Lys Val Leu Leu Leu 65 70 75 80 Gly Ala Gly Asp Ser Gly Lys Thr Thr Ile Met Lys Gln Met Arg Leu 85 90 95 Leu Tyr Ser Pro Gly Phe Ser Gln Val Val Arg Lys Gln Tyr Arg Val 100 105 110 Met Ile Phe Glu Asn Ile Ile Ser Ser Leu Cys Leu Leu Leu Glu Ala 115 120 125 Met Asp Asn Ser Asn Val Ser Leu Leu Pro Glu Asn Glu Lys Tyr Arg 130 135 140 Ala Val Ile Leu Arg Lys His Thr Ser Gln Pro Asn Glu Pro Phe Ser 145 150 155 160 Pro Glu Ile Tyr Glu Ala Val His Ala Leu Thr Leu Asp Thr Lys Leu 165 170 175 Arg Thr Val Gln Ser Cys Gly Thr Asn Leu Ser Leu Leu Asp Asn Phe 180 185 190 Tyr Tyr Tyr Gln Asp His Ile Asp Arg Ile Phe Asp Pro Gln Tyr Ile 195 200 205 Pro Ser Asp Gln Asp Ile Leu His Cys Arg Ile Lys Thr Thr Gly Ile 210 215 220 Ser Glu Glu Thr Phe Leu Leu Asn Arg His His Tyr Arg Phe Phe Asp 225 230 235 240 Val Gly Gly Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu 245 250 255 Asn Val Thr Ala Leu Leu Phe Leu Val Ser Leu Ala Gly Tyr Asp Gln 260 265 270 Cys Leu Val Glu Asp Asn Ser Gly Asn Gln Met Gln Glu Ala Leu Leu 275 280 285 Leu Trp Asp Ser Ile Cys Asn Ser Ser Trp Phe Ser Glu Ser Ala Met 290 295 300 Ile Leu Phe Leu Asn Lys Leu Asp Leu Phe Lys Arg Lys Val His Ile 305 310 315 320 Ser Pro Ile Gln Lys His Phe Pro Asp Tyr Gln Glu Val Gly Ser Thr 325 330 335 Pro Thr Phe Val Gln Thr Gln Cys Pro Leu Ala Asp Asn Ala Val Arg 340 345 350 Ser Gly Met Tyr Tyr Phe Tyr Leu Lys Phe Glu Ser Leu Asn Arg Ile 355 360 365 Ala Ser Arg Ser Cys Tyr Cys His Phe Thr Thr Ala Thr Asp Thr Ser 370 375 380 Leu Leu Gln Arg Val Met Val Ser Val Gln Asp Thr Ile Met Ser Asn 385 390 395 400 Asn Leu Glu Cys Gly Leu Tyr 405 7 407 PRT Artificial Sequence Description of Artificial Sequence G alpha transplant 7 Met Gly Cys Met Ser Ser Lys Tyr Ala Asp Thr Ser Gly Gly Glu Val 1 5 10 15 Ile Gln Lys Lys Leu Ser Asp Thr Gln Thr Ser Asn Ser Ser Thr Thr 20 25 30 Gly Ser Gln Asn Ala Arg Val Pro Val Leu Glu Asn Trp Leu Asn Ile 35 40 45 Val Leu Arg Gly Lys Pro Gln Asn Val Glu Ser Ser Gly Val Arg Val 50 55 60 Lys Gly Asn Ser Thr Ser Gly Gly Asn Asp Ile Lys Val Leu Leu Leu 65 70 75 80 Gly Ala Gly Asp Ser Gly Lys Thr Thr Ile Met Lys Gln Met Arg Leu 85 90 95 Leu Tyr Ser Pro Gly Phe Ser Gln Val Val Arg Lys Gln Tyr Arg Val 100 105 110 Met Ile Phe Glu Asn Ile Ile Ser Ser Leu Cys Leu Leu Leu Glu Ala 115 120 125 Met Asp Asn Ser Asn Val Ser Leu Leu Pro Glu Asn Glu Lys Tyr Arg 130 135 140 Ala Val Ile Leu Arg Lys His Thr Ser Gln Pro Asn Glu Pro Phe Ser 145 150 155 160 Pro Glu Ile Tyr Glu Ala Val His Ala Leu Thr Leu Asp Thr Lys Leu 165 170 175 Arg Thr Val Gln Ser Cys Gly Thr Asn Leu Ser Leu Leu Asp Asn Phe 180 185 190 Tyr Tyr Tyr Gln Asp His Ile Asp Arg Ile Phe Asp Pro Gln Tyr Ile 195 200 205 Pro Ser Asp Gln Asp Ile Leu His Cys Arg Ile Lys Thr Thr Gly Ile 210 215 220 Ser Glu Glu Thr Phe Leu Leu Asn Arg His His Tyr Arg Phe Phe Asp 225 230 235 240 Val Gly Gly Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu 245 250 255 Asn Val Thr Ala Leu Leu Phe Leu Val Ser Leu Ala Gly Tyr Asp Gln 260 265 270 Cys Leu Val Glu Asp Asn Ser Gly Asn Gln Met Gln Glu Ala Leu Leu 275 280 285 Leu Trp Asp Ser Ile Cys Asn Ser Ser Trp Phe Ser Glu Ser Ala Met 290 295 300 Ile Leu Phe Leu Asn Lys Leu Asp Leu Phe Lys Arg Lys Val His Ile 305 310 315 320 Ser Pro Ile Gln Lys His Phe Pro Asp Tyr Gln Glu Val Gly Ser Thr 325 330 335 Pro Thr Phe Val Gln Thr Gln Cys Pro Leu Ala Asp Asn Ala Val Arg 340 345 350 Ser Gly Met Tyr Tyr Phe Tyr Leu Lys Phe Glu Ser Leu Asn Arg Ile 355 360 365 Ala Ser Arg Ser Cys Tyr Cys His Phe Thr Thr Ala Thr Asp Thr Ser 370 375 380 Leu Leu Gln Arg Val Met Val Ser Val Gln Asp Thr Ile Met Ser Asn 385 390 395 400 Asn Leu Tyr Ile Gly Leu Cys 405 8 407 PRT Artificial Sequence Description of Artificial Sequence G alpha transplant 8 Met Gly Cys Met Ser Ser Lys Tyr Ala Asp Thr Ser Gly Gly Glu Val 1 5 10 15 Ile Gln Lys Lys Leu Ser Asp Thr Gln Thr Ser Asn Ser Ser Thr Thr 20 25 30 Gly Ser Gln Asn Ala Arg Val Pro Val Leu Glu Asn Trp Leu Asn Ile 35 40 45 Val Leu Arg Gly Lys Pro Gln Asn Val Glu Ser Ser Gly Val Arg Val 50 55 60 Lys Gly Asn Ser Thr Ser Gly Gly Asn Asp Ile Lys Val Leu Leu Leu 65 70 75 80 Gly Ala Gly Asp Ser Gly Lys Thr Thr Ile Met Lys Gln Met Arg Leu 85 90 95 Leu Tyr Ser Pro Gly Phe Ser Gln Val Val Arg Lys Gln Tyr Arg Val 100 105 110 Met Ile Phe Glu Asn Ile Ile Ser Ser Leu Cys Leu Leu Leu Glu Ala 115 120 125 Met Asp Asn Ser Asn Val Ser Leu Leu Pro Glu Asn Glu Lys Tyr Arg 130 135 140 Ala Val Ile Leu Arg Lys His Thr Ser Gln Pro Asn Glu Pro Phe Ser 145 150 155 160 Pro Glu Ile Tyr Glu Ala Val His Ala Leu Thr Leu Asp Thr Lys Leu 165 170 175 Arg Thr Val Gln Ser Cys Gly Thr Asn Leu Ser Leu Leu Asp Asn Phe 180 185 190 Tyr Tyr Tyr Gln Asp His Ile Asp Arg Ile Phe Asp Pro Gln Tyr Ile 195 200 205 Pro Ser Asp Gln Asp Ile Leu His Cys Arg Ile Lys Thr Thr Gly Ile 210 215 220 Ser Glu Glu Thr Phe Leu Leu Asn Arg His His Tyr Arg Phe Phe Asp 225 230 235 240 Val Gly Gly Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu 245 250 255 Asn Val Thr Ala Leu Leu Phe Leu Val Ser Leu Ala Gly Tyr Asp Gln 260 265 270 Cys Leu Val Glu Asp Asn Ser Gly Asn Gln Met Gln Glu Ala Leu Leu 275 280 285 Leu Trp Asp Ser Ile Cys Asn Ser Ser Trp Phe Ser Glu Ser Ala Met 290 295 300 Ile Leu Phe Leu Asn Lys Leu Asp Leu Phe Lys Arg Lys Val His Ile 305 310 315 320 Ser Pro Ile Gln Lys His Phe Pro Asp Tyr Gln Glu Val Gly Ser Thr 325 330 335 Pro Thr Phe Val Gln Thr Gln Cys Pro Leu Ala Asp Asn Ala Val Arg 340 345 350 Ser Gly Met Tyr Tyr Phe Tyr Leu Lys Phe Glu Ser Leu Asn Arg Ile 355 360 365 Ala Ser Arg Ser Cys Tyr Cys His Phe Thr Thr Ala Thr Asp Thr Ser 370 375 380 Leu Leu Gln Arg Val Met Val Ser Val Gln Asp Thr Ile Met Ser Asn 385 390 395 400 Asn Leu Glu Asn Val Arg Phe 405 9 407 PRT Artificial Sequence Description of Artificial Sequence G alpha transplant 9 Met Gly Cys Met Ser Ser Lys Tyr Ala Asp Thr Ser Gly Gly Glu Val 1 5 10 15 Ile Gln Lys Lys Leu Ser Asp Thr Gln Thr Ser Asn Ser Ser Thr Thr 20 25 30 Gly Ser Gln Asn Ala Arg Val Pro Val Leu Glu Asn Trp Leu Asn Ile 35 40 45 Val Leu Arg Gly Lys Pro Gln Asn Val Glu Ser Ser Gly Val Arg Val 50 55 60 Lys Gly Asn Ser Thr Ser Gly Gly Asn Asp Ile Lys Val Leu Leu Leu 65 70 75 80 Gly Ala Gly Asp Ser Gly Lys Thr Thr Ile Met Lys Gln Met Arg Leu 85 90 95 Leu Tyr Ser Pro Gly Phe Ser Gln Val Val Arg Lys Gln Tyr Arg Val 100 105 110 Met Ile Phe Glu Asn Ile Ile Ser Ser Leu Cys Leu Leu Leu Glu Ala 115 120 125 Met Asp Asn Ser Asn Val Ser Leu Leu Pro Glu Asn Glu Lys Tyr Arg 130 135 140 Ala Val Ile Leu Arg Lys His Thr Ser Gln Pro Asn Glu Pro Phe Ser 145 150 155 160 Pro Glu Ile Tyr Glu Ala Val His Ala Leu Thr Leu Asp Thr Lys Leu 165 170 175 Arg Thr Val Gln Ser Cys Gly Thr Asn Leu Ser Leu Leu Asp Asn Phe 180 185 190 Tyr Tyr Tyr Gln Asp His Ile Asp Arg Ile Phe Asp Pro Gln Tyr Ile 195 200 205 Pro Ser Asp Gln Asp Ile Leu His Cys Arg Ile Lys Thr Thr Gly Ile 210 215 220 Ser Glu Glu Thr Phe Leu Leu Asn Arg His His Tyr Arg Phe Phe Asp 225 230 235 240 Val Gly Gly Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu 245 250 255 Asn Val Thr Ala Leu Leu Phe Leu Val Ser Leu Ala Gly Tyr Asp Gln 260 265 270 Cys Leu Val Glu Asp Asn Ser Gly Asn Gln Met Gln Glu Ala Leu Leu 275 280 285 Leu Trp Asp Ser Ile Cys Asn Ser Ser Trp Phe Ser Glu Ser Ala Met 290 295 300 Ile Leu Phe Leu Asn Lys Leu Asp Leu Phe Lys Arg Lys Val His Ile 305 310 315 320 Ser Pro Ile Gln Lys His Phe Pro Asp Tyr Gln Glu Val Gly Ser Thr 325 330 335 Pro Thr Phe Val Gln Thr Gln Cys Pro Leu Ala Asp Asn Ala Val Arg 340 345 350 Ser Gly Met Tyr Tyr Phe Tyr Leu Lys Phe Glu Ser Leu Asn Arg Ile 355 360 365 Ala Ser Arg Ser Cys Tyr Cys His Phe Thr Thr Ala Thr Asp Thr Ser 370 375 380 Leu Leu Gln Arg Val Met Val Ser Val Gln Asp Thr Ile Met Ser Asn 385 390 395 400 Asn Leu Arg Leu Val Phe Arg 405 10 407 PRT Artificial Sequence Description of Artificial Sequence G alpha transplant 10 Met Gly Cys Met Ser Ser Lys Tyr Ala Asp Thr Ser Gly Gly Glu Val 1 5 10 15 Ile Gln Lys Lys Leu Ser Asp Thr Gln Thr Ser Asn Ser Ser Thr Thr 20 25 30 Gly Ser Gln Asn Ala Arg Val Pro Val Leu Glu Asn Trp Leu Asn Ile 35 40 45 Val Leu Arg Gly Lys Pro Gln Asn Val Glu Ser Ser Gly Val Arg Val 50 55 60 Lys Gly Asn Ser Thr Ser Gly Gly Asn Asp Ile Lys Val Leu Leu Leu 65 70 75 80 Gly Ala Gly Asp Ser Gly Lys Thr Thr Ile Met Lys Gln Met Arg Leu 85 90 95 Leu Tyr Ser Pro Gly Phe Ser Gln Val Val Arg Lys Gln Tyr Arg Val 100 105 110 Met Ile Phe Glu Asn Ile Ile Ser Ser Leu Cys Leu Leu Leu Glu Ala 115 120 125 Met Asp Asn Ser Asn Val Ser Leu Leu Pro Glu Asn Glu Lys Tyr Arg 130 135 140 Ala Val Ile Leu Arg Lys His Thr Ser Gln Pro Asn Glu Pro Phe Ser 145 150 155 160 Pro Glu Ile Tyr Glu Ala Val His Ala Leu Thr Leu Asp Thr Lys Leu 165 170 175 Arg Thr Val Gln Ser Cys Gly Thr Asn Leu Ser Leu Leu Asp Asn Phe 180 185 190 Tyr Tyr Tyr Gln Asp His Ile Asp Arg Ile Phe Asp Pro Gln Tyr Ile 195 200 205 Pro Ser Asp Gln Asp Ile Leu His Cys Arg Ile Lys Thr Thr Gly Ile 210 215 220 Ser Glu Glu Thr Phe Leu Leu Asn Arg His His Tyr Arg Phe Phe Asp 225 230 235 240 Val Gly Gly Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu 245 250 255 Asn Val Thr Ala Leu Leu Phe Leu Val Ser Leu Ala Gly Tyr Asp Gln 260 265 270 Cys Leu Val Glu Asp Asn Ser Gly Asn Gln Met Gln Glu Ala Leu Leu 275 280 285 Leu Trp Asp Ser Ile Cys Asn Ser Ser Trp Phe Ser Glu Ser Ala Met 290 295 300 Ile Leu Phe Leu Asn Lys Leu Asp Leu Phe Lys Arg Lys Val His Ile 305 310 315 320 Ser Pro Ile Gln Lys His Phe Pro Asp Tyr Gln Glu Val Gly Ser Thr 325 330 335 Pro Thr Phe Val Gln Thr Gln Cys Pro Leu Ala Asp Asn Ala Val Arg 340 345 350 Ser Gly Met Tyr Tyr Phe Tyr Leu Lys Phe Glu Ser Leu Asn Arg Ile 355 360 365 Ala Ser Arg Ser Cys Tyr Cys His Phe Thr Thr Ala Thr Asp Thr Ser 370 375 380 Leu Leu Gln Arg Val Met Val Ser Val Gln Asp Thr Ile Met Ser Asn 385 390 395 400 Asn Leu Glu Phe Asn Leu Val 405 11 407 PRT Artificial Sequence Description of Artificial Sequence G alpha transplant 11 Met Gly Cys Met Ser Ser Lys Tyr Ala Asp Thr Ser Gly Gly Glu Val 1 5 10 15 Ile Gln Lys Lys Leu Ser Asp Thr Gln Thr Ser Asn Ser Ser Thr Thr 20 25 30 Gly Ser Gln Asn Ala Arg Val Pro Val Leu Glu Asn Trp Leu Asn Ile 35 40 45 Val Leu Arg Gly Lys Pro Gln Asn Val Glu Ser Ser Gly Val Arg Val 50 55 60 Lys Gly Asn Ser Thr Ser Gly Gly Asn Asp Ile Lys Val Leu Leu Leu 65 70 75 80 Gly Ala Gly Asp Ser Gly Lys Thr Thr Ile Met Lys Gln Met Arg Leu 85 90 95 Leu Tyr Ser Pro Gly Phe Ser Gln Val Val Arg Lys Gln Tyr Arg Val 100 105 110 Met Ile Phe Glu Asn Ile Ile Ser Ser Leu Cys Leu Leu Leu Glu Ala 115 120 125 Met Asp Asn Ser Asn Val Ser Leu Leu Pro Glu Asn Glu Lys Tyr Arg 130 135 140 Ala Val Ile Leu Arg Lys His Thr Ser Gln Pro Asn Glu Pro Phe Ser 145 150 155 160 Pro Glu Ile Tyr Glu Ala Val His Ala Leu Thr Leu Asp Thr Lys Leu 165 170 175 Arg Thr Val Gln Ser Cys Gly Thr Asn Leu Ser Leu Leu Asp Asn Phe 180 185 190 Tyr Tyr Tyr Gln Asp His Ile Asp Arg Ile Phe Asp Pro Gln Tyr Ile 195 200 205 Pro Ser Asp Gln Asp Ile Leu His Cys Arg Ile Lys Thr Thr Gly Ile 210 215 220 Ser Glu Glu Thr Phe Leu Leu Asn Arg His His Tyr Arg Phe Phe Asp 225 230 235 240 Val Gly Gly Gln Arg Ser Glu Arg Arg Lys Trp Ile His Cys Phe Glu 245 250 255 Asn Val Thr Ala Leu Leu Phe Leu Val Ser Leu Ala Gly Tyr Asp Gln 260 265 270 Cys Leu Val Glu Asp Asn Ser Gly Asn Gln Met Gln Glu Ala Leu Leu 275 280 285 Leu Trp Asp Ser Ile Cys Asn Ser Ser Trp Phe Ser Glu Ser Ala Met 290 295 300 Ile Leu Phe Leu Asn Lys Leu Asp Leu Phe Lys Arg Lys Val His Ile 305 310 315 320 Ser Pro Ile Gln Lys His Phe Pro Asp Tyr Gln Glu Val Gly Ser Thr 325 330 335 Pro Thr Phe Val Gln Thr Gln Cys Pro Leu Ala Asp Asn Ala Val Arg 340 345 350 Ser Gly Met Tyr Tyr Phe Tyr Leu Lys Phe Glu Ser Leu Asn Arg Ile 355 360 365 Ala Ser Arg Ser Cys Tyr Cys His Phe Thr Thr Ala Thr Asp Thr Ser 370 375 380 Leu Leu Gln Arg Val Met Val Ser Val Gln Asp Thr Ile Met Ser Asn 385 390 395 400 Asn Leu Phe Lys Asp Val Arg 405 12 201 PRT Schizosaccharomyces pombe 12 Met Lys Ile Thr Ala Val Ile Ala Leu Leu Phe Ser Leu Ala Ala Ala 1 5 10 15 Ser Pro Ile Pro Val Ala Asp Pro Gly Val Val Ser Val Ser Lys Ser 20 25 30 Tyr Ala Asp Phe Leu Arg Val Tyr Gln Ser Trp Asn Thr Phe Ala Asn 35 40 45 Pro Asp Arg Pro Asn Leu Lys Lys Arg Glu Phe Glu Ala Ala Pro Ala 50 55 60 Lys Thr Tyr Ala Asp Phe Leu Arg Ala Tyr Gln Ser Trp Asn Thr Phe 65 70 75 80 Val Asn Pro Asp Arg Pro Asn Leu Lys Lys Arg Glu Phe Glu Ala Ala 85 90 95 Pro Glu Lys Ser Tyr Ala Asp Phe Leu Arg Ala Tyr His Ser Trp Asn 100 105 110 Thr Phe Val Asn Pro Asp Arg Pro Asn Leu Lys Lys Arg Glu Phe Glu 115 120 125 Ala Ala Pro Ala Lys Thr Tyr Ala Asp Phe Leu Arg Ala Tyr Gln Ser 130 135 140 Trp Asn Thr Phe Val Asn Pro Asp Arg Pro Asn Leu Lys Lys Arg Thr 145 150 155 160 Glu Glu Asp Glu Glu Asn Glu Glu Glu Asp Glu Glu Tyr Tyr Arg Phe 165 170 175 Leu Gln Phe Tyr Ile Met Thr Val Pro Glu Asn Ser Thr Ile Thr Asp 180 185 190 Val Asn Ile Thr Ala Lys Phe Glu Ser 195 200 13 99 PRT Schizosaccharomyces pombe 13 Met Lys Ile Thr Ala Val Ile Ala Leu Leu Phe Ser Leu Ala Ala Ala 1 5 10 15 Ser Pro Ile Pro Val Ala Asp Pro Gly Val Val Ser Val Ser Lys Ser 20 25 30 Tyr Ala Asp Phe Leu Arg Val Tyr Gln Ser Trp Asn Thr Phe Ala Asn 35 40 45 Pro Asp Arg Pro Asn Leu Lys Lys Arg Thr Glu Glu Asp Glu Glu Asn 50 55 60 Glu Glu Glu Asp Glu Glu Tyr Tyr Arg Phe Leu Gln Phe Tyr Ile Met 65 70 75 80 Thr Val Pro Glu Asn Ser Thr Ile Thr Asp Val Asn Ile Thr Ala Lys 85 90 95 Phe Glu Ser 14 415 PRT Homo sapiens 14 Met Gly Gly His Pro Gln Leu Arg Leu Val Lys Ala Leu Leu Leu Leu 1 5 10 15 Gly Leu Asn Pro Val Ser Ala Ser Leu Gln Asp Gln His Cys Glu Ser 20 25 30 Leu Ser Leu Ala Ser Asn Ile Ser Gly Leu Gln Cys Asn Ala Ser Val 35 40 45 Asp Leu Ile Gly Thr Cys Trp Pro Arg Ser Pro Ala Gly Gln Leu Val 50 55 60 Val Arg Pro Cys Pro Ala Phe Phe Tyr Gly Val Arg Tyr Asn Thr Thr 65 70 75 80 Asn Asn Gly Tyr Arg Glu Cys Leu Ala Asn Gly Ser Trp Ala Ala Arg 85 90 95 Val Asn Tyr Ser Glu Cys Gln Glu Ile Leu Asn Glu Glu Lys Lys Ser 100 105 110 Lys Val His Tyr His Val Ala Val Ile Ile Asn Tyr Leu Gly His Cys 115 120 125 Ile Ser Leu Val Ala Leu Leu Val Ala Phe Val Leu Phe Leu Arg Leu 130 135 140 Arg Ser Ile Arg Cys Leu Arg Asn Ile Ile His Trp Asn Leu Ile Ser 145 150 155 160 Ala Phe Ile Leu Arg Asn Ala Thr Trp Phe Val Val Gln Leu Thr Met 165 170 175 Ser Pro Glu Val His Gln Ser Asn Val Gly Trp Cys Arg Leu Val Thr 180 185 190 Ala Ala Tyr Asn Tyr Phe His Val Thr Asn Phe Phe Trp Met Phe Gly 195 200 205 Glu Gly Cys Tyr Leu His Thr Ala Ile Val Leu Thr Tyr Ser Thr Asp 210 215 220 Arg Leu Arg Lys Trp Met Phe Ile Cys Ile Gly Trp Gly Val Pro Phe 225 230 235 240 Pro Ile Ile Val Ala Trp Ala Ile Gly Lys Leu Tyr Tyr Asp Asn Glu 245 250 255 Lys Cys Trp Phe Gly Lys Arg Pro Gly Val Tyr Thr Asp Tyr Ile Tyr 260 265 270 Gln Gly Pro Met Ile Leu Val Leu Leu Ile Asn Phe Ile Phe Leu Phe 275 280 285 Asn Ile Val Arg Ile Leu Met Thr Lys Leu Arg Ala Ser Thr Thr Ser 290 295 300 Glu Thr Ile Gln Tyr Arg Lys Ala Val Lys Ala Thr Leu Val Leu Leu 305 310 315 320 Pro Leu Leu Gly Ile Thr Tyr Met Leu Phe Phe Val Asn Pro Gly Glu 325 330 335 Asp Glu Val Ser Arg Val Val Phe Ile Tyr Phe Asn Ser Phe Leu Glu 340 345 350 Ser Phe Gln Gly Phe Phe Val Ser Val Phe Tyr Cys Phe Leu Asn Ser 355 360 365 Glu Val Arg Ser Ala Ile Arg Lys Arg Trp His Arg Trp Gln Asp Lys 370 375 380 His Ser Ile Arg Ala Arg Val Ala Arg Ala Met Ser Ile Pro Thr Ser 385 390 395 400 Pro Thr Arg Val Ser Phe His Ser Ile Lys Gln Ser Thr Ala Val 405 410 415 15 1224 DNA Schizosaccharomyces pombe 15 atgggatgca tgtcgagtaa atacgctgat acatcaggag gagaagtcat tcaaaagaag 60 ctttcagata cgcaaacctc aaacagctct acaactggaa gtcaaaacgc tcgagttcca 120 gtccttgaaa actggcttaa tatcgtcctg cgtggaaaac cacaaaatgt ggaaagttct 180 ggagtacgcg taaaaggaaa ttctacttca ggtggaaatg acattaaagt tttgctctta 240 ggcgccggtg atagtgggaa aacgaccatt atgaagcaga tgagattatt gtatagcccc 300 ggttttagtc aagtagttag aaagcagtat cgagtgatga tttttgaaaa tatcatctcc 360 tctctatgtc ttcttcttga agctatggat aatagtaatg tctctttact tccggaaaat 420 gagaagtatc gggcagttat cctaagaaaa cacacttctc aacccaatga gccattttct 480 ccagaaatat atgaagctgt tcatgccttg acattggata ccaaacttcg tacggtgcaa 540 agttgtggta ccaacctctc tttgttagac aatttttatt actatcaaga tcacattgat 600 cgaatttttg acccacaata tataccttct gatcaagata tccttcactg tcgtatcaag 660 acgaccggta tatcagaaga aacatttctg ttaaatcgtc atcattaccg attttttgat 720 gtaggaggac agagatcaga gcgcagaaaa tggattcatt gctttgaaaa tgtcactgca 780 ttgttgtttc tcgtttcttt ggcaggttac gatcaatgcc ttgtagagga caattcagga 840 aatcagatgc aggaggcgtt attattatgg gattccatat gtaactctag ctggttttca 900 gaatcagcaa tgatactttt tctaaataaa cttgatttat ttaaaagaaa ggttcacatt 960 tcccccatcc agaagcattt tcctgattac caagaagttg gttcaacacc aacattcgta 1020 caaactcaat gccctcttgc cgacaacgca gttcgaagcg gtatgtatta cttttactta 1080 aagtttgaaa gtcttaatcg catcgcttct cgtagttgct attgccattt taccacagct 1140 acagacacta gtttgctcca aagggtaatg gtatccgttc aagatacgat tatgtccaac 1200 aatctacagt cacttatgtt ttag 1224 16 1224 DNA Artificial Sequence Description of Artificial SequenceG alpha transplant 16 atgggatgca tgtcgagtaa atacgctgat acatcaggag gagaagtcat tcaaaagaag 60 ctttcagata cgcaaacctc aaacagctct acaactggaa gtcaaaacgc tcgagttcca 120 gtccttgaaa actggcttaa tatcgtcctg cgtggaaaac cacaaaatgt ggaaagttct 180 ggagtacgcg taaaaggaaa ttctacttca ggtggaaatg acattaaagt tttgctctta 240 ggcgccggtg atagtgggaa aacgaccatt atgaagcaga tgagattatt gtatagcccc 300 ggttttagtc aagtagttag aaagcagtat cgagtgatga tttttgaaaa tatcatctcc 360 tctctatgtc ttcttcttga agctatggat aatagtaatg tctctttact tccggaaaat 420 gagaagtatc gggcagttat cctaagaaaa cacacttctc aacccaatga gccattttct 480 ccagaaatat atgaagctgt tcatgccttg acattggata ccaaacttcg tacggtgcaa 540 agttgtggta ccaacctctc tttgttagac aatttttatt actatcaaga tcacattgat 600 cgaatttttg acccacaata tataccttct gatcaagata tccttcactg tcgtatcaag 660 acgaccggta tatcagaaga aacatttctg ttaaatcgtc atcattaccg attttttgat 720 gtaggaggac agagatcaga gcgcagaaaa tggattcatt gctttgaaaa tgtcactgca 780 ttgttgtttc tcgtttcttt ggcaggttac gatcaatgcc ttgtagagga caattcagga 840 aatcagatgc aggaggcgtt attattatgg gattccatat gtaactctag ctggttttca 900 gaatcagcaa tgatactttt tctaaataaa cttgatttat ttaaaagaaa ggttcacatt 960 tcccccatcc agaagcattt tcctgattac caagaagttg gttcaacacc aacattcgta 1020 caaactcaat gccctcttgc cgacaacgca gttcgaagcg gtatgtatta cttttactta 1080 aagtttgaaa gtcttaatcg catcgcttct cgtagttgct attgccattt taccacagct 1140 acagacacta gtttgctcca aagggtaatg gtatccgttc aagatacgat tatgtccaac 1200 aatctacaat atgaacttct ttag 1224 17 1224 DNA Artificial Sequence Description of Artificial SequenceG alpha transplant 17 atgggatgca tgtcgagtaa atacgctgat acatcaggag gagaagtcat tcaaaagaag 60 ctttcagata cgcaaacctc aaacagctct acaactggaa gtcaaaacgc tcgagttcca 120 gtccttgaaa actggcttaa tatcgtcctg cgtggaaaac cacaaaatgt ggaaagttct 180 ggagtacgcg taaaaggaaa ttctacttca ggtggaaatg acattaaagt tttgctctta 240 ggcgccggtg atagtgggaa aacgaccatt atgaagcaga tgagattatt gtatagcccc 300 ggttttagtc aagtagttag aaagcagtat cgagtgatga tttttgaaaa tatcatctcc 360 tctctatgtc ttcttcttga agctatggat aatagtaatg tctctttact tccggaaaat 420 gagaagtatc gggcagttat cctaagaaaa cacacttctc aacccaatga gccattttct 480 ccagaaatat atgaagctgt tcatgccttg acattggata ccaaacttcg tacggtgcaa 540 agttgtggta ccaacctctc tttgttagac aatttttatt actatcaaga tcacattgat 600 cgaatttttg acccacaata tataccttct gatcaagata tccttcactg tcgtatcaag 660 acgaccggta tatcagaaga aacatttctg ttaaatcgtc atcattaccg attttttgat 720 gtaggaggac agagatcaga gcgcagaaaa tggattcatt gctttgaaaa tgtcactgca 780 ttgttgtttc tcgtttcttt ggcaggttac gatcaatgcc ttgtagagga caattcagga 840 aatcagatgc aggaggcgtt attattatgg gattccatat gtaactctag ctggttttca 900 gaatcagcaa tgatactttt tctaaataaa cttgatttat ttaaaagaaa ggttcacatt 960 tcccccatcc agaagcattt tcctgattac caagaagttg gttcaacacc aacattcgta 1020 caaactcaat gccctcttgc cgacaacgca gttcgaagcg gtatgtatta cttttactta 1080 aagtttgaaa gtcttaatcg catcgcttct cgtagttgct attgccattt taccacagct 1140 acagacacta gtttgctcca aagggtaatg gtatccgttc aagatacgat tatgtccaac 1200 aatctagaat ataatcttgt ttag 1224 18 1224 DNA Artificial Sequence Description of Artificial SequenceG alpha transplant 18 atgggatgca tgtcgagtaa atacgctgat acatcaggag gagaagtcat tcaaaagaag 60 ctttcagata cgcaaacctc aaacagctct acaactggaa gtcaaaacgc tcgagttcca 120 gtccttgaaa actggcttaa tatcgtcctg cgtggaaaac cacaaaatgt ggaaagttct 180 ggagtacgcg taaaaggaaa ttctacttca ggtggaaatg acattaaagt tttgctctta 240 ggcgccggtg atagtgggaa aacgaccatt atgaagcaga tgagattatt gtatagcccc 300 ggttttagtc aagtagttag aaagcagtat cgagtgatga tttttgaaaa tatcatctcc 360 tctctatgtc ttcttcttga agctatggat aatagtaatg tctctttact tccggaaaat 420 gagaagtatc gggcagttat cctaagaaaa cacacttctc aacccaatga gccattttct 480 ccagaaatat atgaagctgt tcatgccttg acattggata ccaaacttcg tacggtgcaa 540 agttgtggta ccaacctctc tttgttagac aatttttatt actatcaaga tcacattgat 600 cgaatttttg acccacaata tataccttct gatcaagata tccttcactg tcgtatcaag 660 acgaccggta tatcagaaga aacatttctg ttaaatcgtc atcattaccg attttttgat 720 gtaggaggac agagatcaga gcgcagaaaa tggattcatt gctttgaaaa tgtcactgca 780 ttgttgtttc tcgtttcttt ggcaggttac gatcaatgcc ttgtagagga caattcagga 840 aatcagatgc aggaggcgtt attattatgg gattccatat gtaactctag ctggttttca 900 gaatcagcaa tgatactttt tctaaataaa cttgatttat ttaaaagaaa ggttcacatt 960 tcccccatcc agaagcattt tcctgattac caagaagttg gttcaacacc aacattcgta 1020 caaactcaat gccctcttgc cgacaacgca gttcgaagcg gtatgtatta cttttactta 1080 aagtttgaaa gtcttaatcg catcgcttct cgtagttgct attgccattt taccacagct 1140 acagacacta gtttgctcca aagggtaatg gtatccgttc aagatacgat tatgtccaac 1200 aatctaggat gcggacttta ttag 1224 19 1224 DNA Artificial Sequence Description of Artificial SequenceG alpha transplant 19 atgggatgca tgtcgagtaa atacgctgat acatcaggag gagaagtcat tcaaaagaag 60 ctttcagata cgcaaacctc aaacagctct acaactggaa gtcaaaacgc tcgagttcca 120 gtccttgaaa actggcttaa tatcgtcctg cgtggaaaac cacaaaatgt ggaaagttct 180 ggagtacgcg taaaaggaaa ttctacttca ggtggaaatg acattaaagt tttgctctta 240 ggcgccggtg atagtgggaa aacgaccatt atgaagcaga tgagattatt gtatagcccc 300 ggttttagtc aagtagttag aaagcagtat cgagtgatga tttttgaaaa tatcatctcc 360 tctctatgtc ttcttcttga agctatggat aatagtaatg tctctttact tccggaaaat 420 gagaagtatc gggcagttat cctaagaaaa cacacttctc aacccaatga gccattttct 480 ccagaaatat atgaagctgt tcatgccttg acattggata ccaaacttcg tacggtgcaa 540 agttgtggta ccaacctctc tttgttagac aatttttatt actatcaaga tcacattgat 600 cgaatttttg acccacaata tataccttct gatcaagata tccttcactg tcgtatcaag 660 acgaccggta tatcagaaga aacatttctg ttaaatcgtc atcattaccg attttttgat 720 gtaggaggac agagatcaga gcgcagaaaa tggattcatt gctttgaaaa tgtcactgca 780 ttgttgtttc tcgtttcttt ggcaggttac gatcaatgcc ttgtagagga caattcagga 840 aatcagatgc aggaggcgtt attattatgg gattccatat gtaactctag ctggttttca 900 gaatcagcaa tgatactttt tctaaataaa cttgatttat ttaaaagaaa ggttcacatt 960 tcccccatcc agaagcattt tcctgattac caagaagttg gttcaacacc aacattcgta 1020 caaactcaat gccctcttgc cgacaacgca gttcgaagcg gtatgtatta cttttactta 1080 aagtttgaaa gtcttaatcg catcgcttct cgtagttgct attgccattt taccacagct 1140 acagacacta gtttgctcca aagggtaatg gtatccgttc aagatacgat tatgtccaac 1200 aatctagatt gcggactttt ttag 1224 20 1224 DNA Artificial Sequence Description of Artificial SequenceG alpha transplant 20 atgggatgca tgtcgagtaa atacgctgat acatcaggag gagaagtcat tcaaaagaag 60 ctttcagata cgcaaacctc aaacagctct acaactggaa gtcaaaacgc tcgagttcca 120 gtccttgaaa actggcttaa tatcgtcctg cgtggaaaac cacaaaatgt ggaaagttct 180 ggagtacgcg taaaaggaaa ttctacttca ggtggaaatg acattaaagt tttgctctta 240 ggcgccggtg atagtgggaa aacgaccatt atgaagcaga tgagattatt gtatagcccc 300 ggttttagtc aagtagttag aaagcagtat cgagtgatga tttttgaaaa tatcatctcc 360 tctctatgtc ttcttcttga agctatggat aatagtaatg tctctttact tccggaaaat 420 gagaagtatc gggcagttat cctaagaaaa cacacttctc aacccaatga gccattttct 480 ccagaaatat atgaagctgt tcatgccttg acattggata ccaaacttcg tacggtgcaa 540 agttgtggta ccaacctctc tttgttagac aatttttatt actatcaaga tcacattgat 600 cgaatttttg acccacaata tataccttct gatcaagata tccttcactg tcgtatcaag 660 acgaccggta tatcagaaga aacatttctg ttaaatcgtc atcattaccg attttttgat 720 gtaggaggac agagatcaga gcgcagaaaa tggattcatt gctttgaaaa tgtcactgca 780 ttgttgtttc tcgtttcttt ggcaggttac gatcaatgcc ttgtagagga caattcagga 840 aatcagatgc aggaggcgtt attattatgg gattccatat gtaactctag ctggttttca 900 gaatcagcaa tgatactttt tctaaataaa cttgatttat ttaaaagaaa ggttcacatt 960 tcccccatcc agaagcattt tcctgattac caagaagttg gttcaacacc aacattcgta 1020 caaactcaat gccctcttgc cgacaacgca gttcgaagcg gtatgtatta cttttactta 1080 aagtttgaaa gtcttaatcg catcgcttct cgtagttgct attgccattt taccacagct 1140 acagacacta gtttgctcca aagggtaatg gtatccgttc aagatacgat tatgtccaac 1200 aatctagaat gcggacttta ttag 1224 21 1224 DNA Artificial Sequence Description of Artificial SequenceG alpha transplant 21 atgggatgca tgtcgagtaa atacgctgat acatcaggag gagaagtcat tcaaaagaag 60 ctttcagata cgcaaacctc aaacagctct acaactggaa gtcaaaacgc tcgagttcca 120 gtccttgaaa actggcttaa tatcgtcctg cgtggaaaac cacaaaatgt ggaaagttct 180 ggagtacgcg taaaaggaaa ttctacttca ggtggaaatg acattaaagt tttgctctta 240 ggcgccggtg atagtgggaa aacgaccatt atgaagcaga tgagattatt gtatagcccc 300 ggttttagtc aagtagttag aaagcagtat cgagtgatga tttttgaaaa tatcatctcc 360 tctctatgtc ttcttcttga agctatggat aatagtaatg tctctttact tccggaaaat 420 gagaagtatc gggcagttat cctaagaaaa cacacttctc aacccaatga gccattttct 480 ccagaaatat atgaagctgt tcatgccttg acattggata ccaaacttcg tacggtgcaa 540 agttgtggta ccaacctctc tttgttagac aatttttatt actatcaaga tcacattgat 600 cgaatttttg acccacaata tataccttct gatcaagata tccttcactg tcgtatcaag 660 acgaccggta tatcagaaga aacatttctg ttaaatcgtc atcattaccg attttttgat 720 gtaggaggac agagatcaga gcgcagaaaa tggattcatt gctttgaaaa tgtcactgca 780 ttgttgtttc tcgtttcttt ggcaggttac gatcaatgcc ttgtagagga caattcagga 840 aatcagatgc aggaggcgtt attattatgg gattccatat gtaactctag ctggttttca 900 gaatcagcaa tgatactttt tctaaataaa cttgatttat ttaaaagaaa ggttcacatt 960 tcccccatcc agaagcattt tcctgattac caagaagttg gttcaacacc aacattcgta 1020 caaactcaat gccctcttgc cgacaacgca gttcgaagcg gtatgtatta cttttactta 1080 aagtttgaaa gtcttaatcg catcgcttct cgtagttgct attgccattt taccacagct 1140 acagacacta gtttgctcca aagggtaatg gtatccgttc aagatacgat tatgtccaac 1200 aatctatata ttggactttg ctag 1224 22 1224 DNA Artificial Sequence Description of Artificial SequenceG alpha transplant 22 atgggatgca tgtcgagtaa atacgctgat acatcaggag gagaagtcat tcaaaagaag 60 ctttcagata cgcaaacctc aaacagctct acaactggaa gtcaaaacgc tcgagttcca 120 gtccttgaaa actggcttaa tatcgtcctg cgtggaaaac cacaaaatgt ggaaagttct 180 ggagtacgcg taaaaggaaa ttctacttca ggtggaaatg acattaaagt tttgctctta 240 ggcgccggtg atagtgggaa aacgaccatt atgaagcaga tgagattatt gtatagcccc 300 ggttttagtc aagtagttag aaagcagtat cgagtgatga tttttgaaaa tatcatctcc 360 tctctatgtc ttcttcttga agctatggat aatagtaatg tctctttact tccggaaaat 420 gagaagtatc gggcagttat cctaagaaaa cacacttctc aacccaatga gccattttct 480 ccagaaatat atgaagctgt tcatgccttg acattggata ccaaacttcg tacggtgcaa 540 agttgtggta ccaacctctc tttgttagac aatttttatt actatcaaga tcacattgat 600 cgaatttttg acccacaata tataccttct gatcaagata tccttcactg tcgtatcaag 660 acgaccggta tatcagaaga aacatttctg ttaaatcgtc atcattaccg attttttgat 720 gtaggaggac agagatcaga gcgcagaaaa tggattcatt gctttgaaaa tgtcactgca 780 ttgttgtttc tcgtttcttt ggcaggttac gatcaatgcc ttgtagagga caattcagga 840 aatcagatgc aggaggcgtt attattatgg gattccatat gtaactctag ctggttttca 900 gaatcagcaa tgatactttt tctaaataaa cttgatttat ttaaaagaaa ggttcacatt 960 tcccccatcc agaagcattt tcctgattac caagaagttg gttcaacacc aacattcgta 1020 caaactcaat gccctcttgc cgacaacgca gttcgaagcg gtatgtatta cttttactta 1080 aagtttgaaa gtcttaatcg catcgcttct cgtagttgct attgccattt taccacagct 1140 acagacacta gtttgctcca aagggtaatg gtatccgttc aagatacgat tatgtccaac 1200 aatctagata ttatgcttca atag 1224 23 1224 DNA Artificial Sequence Description of Artificial SequenceG alpha transplant 23 atgggatgca tgtcgagtaa atacgctgat acatcaggag gagaagtcat tcaaaagaag 60 ctttcagata cgcaaacctc aaacagctct acaactggaa gtcaaaacgc tcgagttcca 120 gtccttgaaa actggcttaa tatcgtcctg cgtggaaaac cacaaaatgt ggaaagttct 180 ggagtacgcg taaaaggaaa ttctacttca ggtggaaatg acattaaagt tttgctctta 240 ggcgccggtg atagtgggaa aacgaccatt atgaagcaga tgagattatt gtatagcccc 300 ggttttagtc aagtagttag aaagcagtat cgagtgatga tttttgaaaa tatcatctcc 360 tctctatgtc ttcttcttga agctatggat aatagtaatg tctctttact tccggaaaat 420 gagaagtatc gggcagttat cctaagaaaa cacacttctc aacccaatga gccattttct 480 ccagaaatat atgaagctgt tcatgccttg acattggata ccaaacttcg tacggtgcaa 540 agttgtggta ccaacctctc tttgttagac aatttttatt actatcaaga tcacattgat 600 cgaatttttg acccacaata tataccttct gatcaagata tccttcactg tcgtatcaag 660 acgaccggta tatcagaaga aacatttctg ttaaatcgtc atcattaccg attttttgat 720 gtaggaggac agagatcaga gcgcagaaaa tggattcatt gctttgaaaa tgtcactgca 780 ttgttgtttc tcgtttcttt ggcaggttac gatcaatgcc ttgtagagga caattcagga 840 aatcagatgc aggaggcgtt attattatgg gattccatat gtaactctag ctggttttca 900 gaatcagcaa tgatactttt tctaaataaa cttgatttat ttaaaagaaa ggttcacatt 960 tcccccatcc agaagcattt tcctgattac caagaagttg gttcaacacc aacattcgta 1020 caaactcaat gccctcttgc cgacaacgca gttcgaagcg gtatgtatta cttttactta 1080 aagtttgaaa gtcttaatcg catcgcttct cgtagttgct attgccattt taccacagct 1140 acagacacta gtttgctcca aagggtaatg gtatccgttc aagatacgat tatgtccaac 1200 aatctacaac ttatgcttca atag 1224 24 1224 DNA Artificial Sequence Description of Artificial SequenceG alpha transplant 24 atgggatgca tgtcgagtaa atacgctgat acatcaggag gagaagtcat tcaaaagaag 60 ctttcagata cgcaaacctc aaacagctct acaactggaa gtcaaaacgc tcgagttcca 120 gtccttgaaa actggcttaa tatcgtcctg cgtggaaaac cacaaaatgt ggaaagttct 180 ggagtacgcg taaaaggaaa ttctacttca ggtggaaatg acattaaagt tttgctctta 240 ggcgccggtg atagtgggaa aacgaccatt atgaagcaga tgagattatt gtatagcccc 300 ggttttagtc aagtagttag aaagcagtat cgagtgatga tttttgaaaa tatcatctcc 360 tctctatgtc ttcttcttga agctatggat aatagtaatg tctctttact tccggaaaat 420 gagaagtatc gggcagttat cctaagaaaa cacacttctc aacccaatga gccattttct 480 ccagaaatat atgaagctgt tcatgccttg acattggata ccaaacttcg tacggtgcaa 540 agttgtggta ccaacctctc tttgttagac aatttttatt actatcaaga tcacattgat 600 cgaatttttg acccacaata tataccttct gatcaagata tccttcactg tcgtatcaag 660 acgaccggta tatcagaaga aacatttctg ttaaatcgtc atcattaccg attttttgat 720 gtaggaggac agagatcaga gcgcagaaaa tggattcatt gctttgaaaa tgtcactgca 780 ttgttgtttc tcgtttcttt ggcaggttac gatcaatgcc ttgtagagga caattcagga 840 aatcagatgc aggaggcgtt attattatgg gattccatat gtaactctag ctggttttca 900 gaatcagcaa tgatactttt tctaaataaa cttgatttat ttaaaagaaa ggttcacatt 960 tcccccatcc agaagcattt tcctgattac caagaagttg gttcaacacc aacattcgta 1020 caaactcaat gccctcttgc cgacaacgca gttcgaagcg gtatgtatta cttttactta 1080 aagtttgaaa gtcttaatcg catcgcttct cgtagttgct attgccattt taccacagct 1140 acagacacta gtttgctcca aagggtaatg gtatccgttc aagatacgat tatgtccaac 1200 aatctagaat ttaatcttgt ttag 1224 25 1224 DNA Artificial Sequence Description of Artificial SequenceG alpha transplant 25 atgggatgca tgtcgagtaa atacgctgat acatcaggag gagaagtcat tcaaaagaag 60 ctttcagata cgcaaacctc aaacagctct acaactggaa gtcaaaacgc tcgagttcca 120 gtccttgaaa actggcttaa tatcgtcctg cgtggaaaac cacaaaatgt ggaaagttct 180 ggagtacgcg taaaaggaaa ttctacttca ggtggaaatg acattaaagt tttgctctta 240 ggcgccggtg atagtgggaa aacgaccatt atgaagcaga tgagattatt gtatagcccc 300 ggttttagtc aagtagttag aaagcagtat cgagtgatga tttttgaaaa tatcatctcc 360 tctctatgtc ttcttcttga agctatggat aatagtaatg tctctttact tccggaaaat 420 gagaagtatc gggcagttat cctaagaaaa cacacttctc aacccaatga gccattttct 480 ccagaaatat atgaagctgt tcatgccttg acattggata ccaaacttcg tacggtgcaa 540 agttgtggta ccaacctctc tttgttagac aatttttatt actatcaaga tcacattgat 600 cgaatttttg acccacaata tataccttct gatcaagata tccttcactg tcgtatcaag 660 acgaccggta tatcagaaga aacatttctg ttaaatcgtc atcattaccg attttttgat 720 gtaggaggac agagatcaga gcgcagaaaa tggattcatt gctttgaaaa tgtcactgca 780 ttgttgtttc tcgtttcttt ggcaggttac gatcaatgcc ttgtagagga caattcagga 840 aatcagatgc aggaggcgtt attattatgg gattccatat gtaactctag ctggttttca 900 gaatcagcaa tgatactttt tctaaataaa cttgatttat ttaaaagaaa ggttcacatt 960 tcccccatcc agaagcattt tcctgattac caagaagttg gttcaacacc aacattcgta 1020 caaactcaat gccctcttgc cgacaacgca gttcgaagcg gtatgtatta cttttactta 1080 aagtttgaaa gtcttaatcg catcgcttct cgtagttgct attgccattt taccacagct 1140 acagacacta gtttgctcca aagggtaatg gtatccgttc aagatacgat tatgtccaac 1200 aatctagaaa ttaatcttct ttag 1224 26 625 DNA Schizosaccharomyces pombe 26 ggggatccac catgaagatc accgctgtca ttgccctttt attctcactt gctgctgcct 60 cacctattcc agttgccgat cctggtgtgg tttcagttag caagtcatat gctgatttcc 120 ttcgtgttta ccaaagttgg aacacttttg ctaatcctga tagacccaac ttgaaaaagc 180 gcgaattcga agctgctccc gcaaaaactt atgctgattt ccttcgtgct tatcaaagtt 240 ggaacacttt tgttaatcct gacagaccca atttgaaaaa gcgtgagttt gaagctgccc 300 cagagaagag ttatgctgat ttccttcgtg cttaccatag ttggaacact tttgttaatc 360 ctgacagacc caacttgaaa aagcgcgaat tcgaagctgc tcccgcaaaa acttatgctg 420 atttccttcg tgcttaccaa agttggaaca cttttgttaa tcctgacaga cccaacttga 480 aaaagcgcac tgaagaagat gaagagaatg aggaagagga tgaagaatac tatcgctttc 540 ttcagtttta tatcatgact gtcccagaga attccactat tacagatgtc aatattactg 600 ccaaatttga gagctaagga tcccc 625 27 319 DNA Schizosaccharomyces pombe 27 ggggatccac catgaagatc accgctgtca ttgccctttt attctcactt gctgctgcct 60 cacctattcc agttgccgat cctggtgtgg tttcagttag caagtcatat gctgatttcc 120 ttcgtgttta ccaaagttgg aacacttttg ctaatcctga tagacccaac ttgaaaaagc 180 gcactgaaga agatgaagag aatgaggaag aggatgaaga atactatcgc tttcttcagt 240 tttatatcat gactgtccca gagaattcca ctattacaga tgtcaatatt actgccaaat 300 ttgagagcta aggatcccc 319 28 1248 DNA Homo sapiens 28 atgggagggc acccgcagct ccgtctcgtc aaggcccttc tccttctggg gctgaacccc 60 gtctctgcct ccctccagga ccagcactgc gagagcctgt ccctggccag caacatctca 120 29 8800 DNA Artificial Sequence Description of Artificial SequencepREP3Xr 29 aagcttgcat gcctgcaggt cgatcgactc tagaggatca gaaaattatc gccataaaag 60 acagaataag tcatcagcgg ttgtttcatt tcctatattt tttttttatt tttttatttt 120 ttaataaggg aaaatttaac gtctaaggat acagaagatt gttagcacat taaagtaata 180 aaggcttaag tagtaagtgc cttagcatgt tattgtattt caaaggacat aatctaaaat 240 aataacaata tcatttctca caagttattc aattttcttt tttttttcta ataatatcaa 300 gaatgtatta tttgtttgac ataagtcaac taatttattt aatatgctgg attaatcttg 360 cagacatgta aattaacaag ttttagtcaa ataacgttga agtttcaatg aactcaaata 420 atttctcttt ttttttatat aaccatatgt ctaatctgat ttatattttc cgcagggatc 480 aactgaagtt atgacatttg gattggatca cttataacct tggtcgccaa ataatacaaa 540 aatcagcgtt ataaaacaaa gaaggttttt gttaagaaat taatcctctt tcttgataag 600 aaagttgaac cgaaattgca gatactgata tatgaaaata atacccacaa ttttgggaat 660 agcgcaagcc tcaatttaaa caataggtga ggacacatga taatgacctc aatgattgtt 720 agaagaaaag agcctcatta caaaatcgaa aaatgaatgg ttgggtacaa gtttccaaaa 780 catggtaaag tggactttgc gtatgagacg taaatagaaa aaaacacttg ttatatgttt 840 tctagaatta ttgttgtctc tttatggttg gatgatgcaa aatagtaatt tcggttagtt 900 gctgtaaaac accacgagac aaatagatat ggatatttat taaatcagga aaaacgtaac 960 tctcggctac tggatggttc agtcacccaa cgattactgg ggagagaaaa cagggcaaaa 1020 gcaaagctta aaggaatccg attgtcattc ggcaatgtgc agcgaaacta aaaaccggat 1080 aatggacctg ttaatcgaaa cattgaagat atataaagga agaggaatcc tggcatatca 1140 tcaattgaat aagttgaatt aattatttca atctcattct cactttctga cttatagtcg 1200 ctttgttaaa ttggcctcga ggtcgactct agagatatcg gatccccggg taaaaggaat 1260 gtctcccttg ccagtactgc tagggttttt ctttcaaact atggaagccc attcaagctg 1320 catattacga ttttgttttt cgcttttaga aagtggttta gatgagataa tagaaaaatt 1380 cttgatctcc gacaacgagt acttttattt tttttgctaa tcactttact caatattagc 1440 tcgaaatcgt agaaacgtag acgggtgcgg gataccgagt ggtgtagtta agaattttta 1500 taaaccacgt ggcccaaaaa tatgaaccca aaacgtttat acatgagtat actttaagaa 1560 ggctataccc cttcgtgtta gatgtagttt tagctaccca acccgagtct atgagcttga 1620 cttcagatgt agaaggcatt aaatcgtttt gaatattaat taaaaaacga tgaaaattaa 1680 atatttaaaa gcaatcatac gctgaaaatt tagtgctgtg gctaatcctt caacatggaa 1740 atgccataaa agtgactttg acaaaaaaaa aagtatatac aggtagtaaa ctcatctact 1800 tcattgactt tgtttacagc atgtggaagg aggaatattt attgctaaat cgtagtttaa 1860 cattcaataa gtaatactat tgaaattcga caagattggc cgcatggatg aaaaagaggc 1920 attttgcttt gggagaatta gttcaaatta gaactgaaaa aaaaaacttt acgaggcaaa 1980 aatgtcggat tgagatcgta aaagttcgct cgtcgtcttt tgctttgtga ttgttttcat 2040 ggatacatct tgctggatat ttaaatttta gtactatgta taagatattc tataaatgtt 2100 ttatcaccca aacctgttag cgccttctta attctattca atctggcttt tgctctgaga 2160 ctacttcttg gactttcact acttgttagt tatacggaat ttgtgtaatt agaagtgaaa 2220 taatcctttc tattagtaat gcgagctcga attcgagtct aactccttaa ccactcggac 2280 atagtgactt atctgacact cattgtaaat taatatatat ataggcattt tgtttagtta 2340 aaggtactta agtaattagt ataaacgaac caattttata atcaggaagt taagtgaatg 2400 gtagcacatg tcgtaaaaat tgtgaatttt tattgaataa tattttaaat acaagccttt 2460 ctagactagg tatactcata aacatatatg agcaaaagga tagaggagat tacattgcat 2520 cttctacaaa tttatttatt gccctttact gaaaaattaa ataatgagta ctaaatgata 2580 aaaagcgctc agtacaagaa agattgcaaa aatattgcat tcttcatgaa ttaaagttgc 2640 atataaggca tattgaaagt aatagtacta aaacagcagt tagcgaaaat taatagaatt 2700 atattcgcaa gacaattgtg acattaaatt aaaaaattgt agaattttta ctatcctctt 2760 taacgccatg agcctttata aaaaggttaa attagtttta acattctttt tttgagtaag 2820 agtttacaat ttatcaaaac ctgtgttatt atattcatta gtttcaattt attagcatct 2880 agagaaaaat caattggcag ttacattgtt ggaatttatg aagaaaaaga atctacaacg 2940 gagaataagt tgctgatcgc tttccctaaa attgtatatt ttgctgagct tattttgaca 3000 tttcgttgaa gttttctcta attcgcattc attttaagta aaacaatgag aaataaaatt 3060 acaaaaaata caaattaaaa tacaattttt agctataatt atagacgatg cccttgtatc 3120 ccattctgtc tcgcttgccc ctacttttta tcttttatat accataatga acgctgccgc 3180 tactaaccat accccgattt tacatttcgg actcccaagg acgtacaaaa tagaaaacta 3240 tagaaaaaaa taatcagaaa atagcatgtc atctctttgt aaaacgcgtt tgcaagaaga 3300 aaggaaacaa tggagaagag atcatccatt tgtatgtaaa ttttagtaaa cttgaagaaa 3360 tcactaacaa cttctcttac ttagggattc tatgcaaaac cttgtaaatc atctgatgga 3420 ggactcgatt taatgaattg gaaggttgga attcactggc cgtcgtttta caacgtcgtg 3480 actgggaaaa ccctggcgtt acccaactta atcgccttgc agcacatccc cctttcgcca 3540 gctggcgtaa tagcgaagag gcccgcaccg atcgcccttc ccaacagttg cgcagcctga 3600 atggcgaatg gcgcctgatg cggtattttc tccttacgca tctgtgcggt atttcacacc 3660 gcatacgtca aagcaaccat agtacgcgcc ctgtagcggc gcattaagcg cggcgggtgt 3720 ggtggttacg cgcagcgtga ccgctacact tgccagcgcc ttagcgcccg ctcctttcgc 3780 tttcttccct tcctttctcg ccacgttcgc cggctttccc cgtcaagctc taaatcgggg 3840 gctcccttta gggttccgat ttagtgcttt acggcacctc gaccccaaaa aacttgattt 3900 gggtgatggt tcacgtagtg ggacatcgcc ctgatagacg gtttttcgcc ctttgacgtt 3960 ggagtccacg ttctttaata gtggactctt gttccaaact ggaacaacac tcaactctat 4020 ctcgggctat tcttttgatt tataagggat tttgccgatt tcggtctatt ggttaaaaaa 4080 tgagctgatt taacaaaaat ttaacgcgaa ttttaacaaa atattaacgt ttacaatttt 4140 atggtgcact ctcagtacaa tctgctctga tgccgcatag ttaagccagc cccgacaccc 4200 gccaacaccc gctgacgcgc cctgacgggc ttgtctgctc ccggcatccg cttacagaca 4260 agctgtgacc gtctccggga gctgcatgtg tcagaggttt tcaccgtcat caccgaaacg 4320 cgcgagacga aagggcctcg tgatacgcct atttttatag gttaatgtca tgataataat 4380 ggtttcttag acgtcaggtg gcacttttcg gggaaatgtg cgcggaaccc ctatttgttt 4440 atttttctaa atacattcaa atatgtatcc gctcatgaga caataaccct gataaatgct 4500 tcaataatat tgaaaaagga agagtatgag tattcaacat ttccgtgtcg cccttattcc 4560 cttttttgcg gcattttgcc ttcctgtttt tgctcaccca gaaacgctgg tgaaagtaaa 4620 agatgctgaa gatcagttgg gtgcacgagt gggttacatc gaactggatc tcaacagcgg 4680 taagatcctt gagagttttc gccccgaaga acgttttcca atgatgagca cttttaaagt 4740 tctgctatgt ggcgcggtat tatcccgtat tgacgccggg caagagcaac tcggtcgccg 4800 catacactat tctcagaatg acttggttga gtactcacca gtcacagaaa agcatcttac 4860 ggatggcatg acagtaagag aattatgcag tgctgccata accatgagtg ataacactgc 4920 ggccaactta cttctgacaa cgatcggagg accgaaggag ctaaccgctt ttttgcacaa 4980 catgggggat catgtaactc gccttgatcg ttgggaaccg gagctgaatg aagccatacc 5040 aaacgacgag cgtgacacca cgatgcctgt agcaatggca acaacgttgc gcaaactatt 5100 aactggcgaa ctacttactc tagcttcccg gcaacaatta atagactgga tggaggcgga 5160 taaagttgca ggaccacttc tgcgctcggc ccttccggct ggctggttta ttgctgataa 5220 atctggagcc ggtgagcgtg ggtctcgcgg tatcattgca gcactggggc cagatggtaa 5280 gccctcccgt atcgtagtta tctacacgac ggggagtcag gcaactatgg atgaacgaaa 5340 tagacagatc gctgagatag gtgcctcact gattaagcat tggtaactgt cagaccaagt 5400 ttactcatat atactttaga ttgatttaaa acttcatttt taatttaaaa ggatctaggt 5460 gaagatcctt tttgataatc tcatgaccaa aatcccttaa cgtgagtttt cgttccactg 5520 agcgtcagac cccgtagaaa agatcaaagg atcttcttga gatccttttt ttctgcgcgt 5580 aatctgctgc ttgcaaacaa aaaaaccacc gctaccagcg gtggtttgtt tgccggatca 5640 agagctacca actctttttc cgaaggtaac tggcttcagc agagcgcaga taccaaatac 5700 tgtccttcta gtgtagccgt agttaggcca ccacttcaag aactctgtag caccgcctac 5760 atacctcgct ctgctaatcc tgttaccagt ggctgctgcc agtggcgata agtcgtgtct 5820 taccgggttg gactcaagac gatagttacc ggataaggcg cagcggtcgg gctgaacggg 5880 gggttcgtgc acacagccca gcttggagcg aacgacctac accgaactga gatacctaca 5940 gcgtgagcta tgagaaagcg ccacgcttcc cgaagggaga aaggcggaca ggtatccggt 6000 aagcggcagg gtcggaacag gagagcgcac gagggagctt ccagggggaa acgcctggta 6060 tctttatagt cctgtcgggt ttcgccacct ctgacttgag cgtcgatttt tgtgatgctc 6120 gtcagggggg cggagcctat ggaaaaacgc cagcaacgcg gcctttttac ggttcctggc 6180 cttttgctgg ccttttgctc acatgttctt tcctgcgtta tcccctgatt ctgtggataa 6240 ccgtattacc gcctttgagt gagctgatac cgctcgccgc agccgaacga ccgagcgcag 6300 cgagtcagtg agcgaggaag cggaagagcg cccaatacgc aaaccgcctc tccccgcgcg 6360 ttggccgatt cattaatgca gctggcacga caggtttccc gactggaaag cgggcagtga 6420 gcgcaacgca attaatgtga gttagctcac tcattaggca ccccaggctt tacactttat 6480 gcttccggct cgtatgttgt gtggaattgt gagcggataa caatttcaca caggaaacag 6540 ctatgaccat gattacgcca agcttgtcga tcgactacgt cgttaaggcc gtttctgaca 6600 gagtaaaatt cttgagggaa ctttcaccat tatgggaaat ggttcaagaa ggtattgact 6660 taaactccat caaatggtca ggtcattgag tgttttttat ttgttgtatt tttttttttt 6720 tagagaaaat cctccaatat caaattagga atcgtagttt catgattttc tgttacacct 6780 aactttttgt gtggtgccct cctccttgtc aatattaatg ttaaagtgca attctttttc 6840 cttatcacgt tgagccatta gtatcaattt gcttacctgt attcctttac tatcctcctt 6900 tttctccttc ttgataaatg tatgtagatt gcgtatatag tttcgtctac cctatgaaca 6960 tattccattt tgtaatttcg tgtcgtttct attatgaatt tcatttataa agtttatgta 7020 caaatatcat aaaaaaagag aatcttttta agcaaggatt ttcttaactt cttcggcgac 7080 agcatcaccg acttcggtgg tactgttgga accacctaaa tcaccagttc tgatacctgc 7140 atccaaaacc tttttaactg catcttcaat ggccttacct tcttcaggca agttcaatga 7200 caatttcaac atcattgcag cagacaagat agtggcgata gggtcaacct tattctttgg 7260 caaatctgga gcagaaccgt ggcatggttc gtacaaacca aatgcggtgt tcttgtctgg 7320 caaagaggcc aaggacgcag atggcaacaa acccaaggaa cctgggataa cggaggcttc 7380 atcggagatg atgtcaccaa acatgttgct ggtgattata ataccattta ggtgggttgg 7440 gttcttaact aggatcatgg cggcagaatc aatcaattga tgttgaacct tcaatgtagg 7500 gaattcgttc ttgatggttt cctccacagt ttttctccat aatcttgaag aggccaaaac 7560 attagcttta tccaaggacc aaataggcaa tggtggctca tgttgtaggg ccatgaaagc 7620 ggccattctt gtgattcttt gcacttctgg aacggtgtat tgttcactat cccaagcgac 7680 accatcacca tcgtcttcct ttctcttacc aaagtaaata cctcccacta attctctgac 7740 aacaacgaag tcagtacctt tagcaaattg tggcttgatt ggagataagt ctaaaagaga 7800 gtcggatgca aagttacatg gtcttaagtt ggcgtacaat tgaagttctt tacggatttt 7860 tagtaaacct tgttcaggtc taacactacc ggtaccccat ttaggaccac ccacagcacc 7920 taacaaaacg gcatcaacct tcttggaggc ttccagcgcc tcatctggaa gtgggacacc 7980 tgtagcatcg atagcagcac caccaattaa atgattttcg aaatcgaact tgacattgga 8040 acgaacatca gaaatagctt taagaacctt aatggcttcg gctgtgattt cttgaccaac 8100 gtggtcacct ggcaaaacga cgatcttctt aggggcagac ataggggcag acattagaat 8160 ggtatatcct tgaaatatat atatatattg ctgaaatgta aaaggtaaga aaagttagaa 8220 agtaagacga ttgctaacca cctattggaa aaaacaatag gtccttaaat aatattgtca 8280 acttcaagta ttgtgatgca agcatttagt catgaacgct tctctattct atatgaaaag 8340 ccggttccgg cctctcacct ttcctttttc tcccaatttt tcagttgaaa aaggtatatg 8400 cgtcaggcga cctctgaaat taacaaaaaa tttccagtca tcgaatttga ttctgtgcga 8460 tagcgcccct gtgtgttctc gttatgttga ggaaaaaaat aatggttgct aagagattcg 8520 aactcttgca tcttacgata cctgagtatt cccacagtta actgcggtca agatatttct 8580 tgaatcaggc gccttagacc gctcggccaa acaaccaatt acttgttgag aaatagagta 8640 taattatcct ataaatataa cgtttttgaa cacacatgaa caaggaagta caggacaatt 8700 gattttgaag agaatgtgga ttttgatgta attgttggga ttccattttt aataaggcaa 8760 taatattagg tatgtagata tactagaagt tctcctcgac 8800 30 10042 DNA Artificial Sequence Description of Artificial SequencepREPXr-CRHR 30 aagcttgcat gcctgcaggt cgatcgactc tagaggatca gaaaattatc gccataaaag 60 acagaataag tcatcagcgg ttgtttcatt tcctatattt tttttttatt tttttatttt 120 ttaataaggg aaaatttaac gtctaaggat acagaagatt gttagcacat taaagtaata 180 aaggcttaag tagtaagtgc cttagcatgt tattgtattt caaaggacat aatctaaaat 240 aataacaata tcatttctca caagttattc aattttcttt tttttttcta ataatatcaa 300 gaatgtatta tttgtttgac ataagtcaac taatttattt aatatgctgg attaatcttg 360 cagacatgta aattaacaag ttttagtcaa ataacgttga agtttcaatg aactcaaata 420 atttctcttt ttttttatat aaccatatgt ctaatctgat ttatattttc cgcagggatc 480 aactgaagtt atgacatttg gattggatca cttataacct tggtcgccaa ataatacaaa 540 aatcagcgtt ataaaacaaa gaaggttttt gttaagaaat taatcctctt tcttgataag 600 aaagttgaac cgaaattgca gatactgata tatgaaaata atacccacaa ttttgggaat 660 agcgcaagcc tcaatttaaa caataggtga ggacacatga taatgacctc aatgattgtt 720 agaagaaaag agcctcatta caaaatcgaa aaatgaatgg ttgggtacaa gtttccaaaa 780 catggtaaag tggactttgc gtatgagacg taaatagaaa aaaacacttg ttatatgttt 840 tctagaatta ttgttgtctc tttatggttg gatgatgcaa aatagtaatt tcggttagtt 900 gctgtaaaac accacgagac aaatagatat ggatatttat taaatcagga aaaacgtaac 960 tctcggctac tggatggttc agtcacccaa cgattactgg ggagagaaaa cagggcaaaa 1020 gcaaagctta aaggaatccg attgtcattc ggcaatgtgc agcgaaacta aaaaccggat 1080 aatggacctg ttaatcgaaa cattgaagat atataaagga agaggaatcc tggcatatca 1140 tcaattgaat aagttgaatt aattatttca atctcattct cactttctga cttatagtcg 1200 ctttgttaaa ttggcctcga ggtcgactct agaatgggag ggcacccgca gctccgtctc 1260 gtcaaggccc ttctccttct ggggctgaac cccgtctctg cctccctcca ggaccagcac 1320 tgcgagagcc tgtccctggc cagcaacatc tcaggactgc agtgcaacgc atccgtggac 1380 ctcattggca cctgctggcc ccgcagccct gcggggcagc tagtggttcg gccctgccct 1440 gcctttttct atggtgtccg ctacaatacc acaaacaatg gctaccggga gtgcctggcc 1500 aatggcagct gggccgcccg cgtgaattac tccgagtgcc aggagatcct caatgaggag 1560 aaaaaaagca aggtgcacta ccatgtcgca gtcatcatca actacctggg ccactgtatc 1620 tccctggtgg ccctcctggt ggcctttgtc ctctttctgc ggctcaggag catccggtgc 1680 ctgcgaaaca tcatccactg gaacctcatc tccgccttca tcctgcgcaa cgccacctgg 1740 ttcgtggtcc agctaaccat gagccccgag gtccaccaga gcaacgtggg ctggtgcagg 1800 ttggtgacag ccgcctacaa ctacttccat gtgaccaact tcttctggat gttcggcgag 1860 ggctgctacc tgcacacagc catcgtgctc acctactcca ctgaccggct gcgcaaatgg 1920 atgttcatct gcattggctg gggtgtgccc ttccccatca ttgtggcctg ggccattggg 1980 aagctgtact acgacaatga gaagtgctgg tttggcaaaa ggcctggggt gtacaccgac 2040 tacatctacc agggccccat gatcctggtc ctgctgatca atttcatctt ccttttcaac 2100 atcgtccgca tcctcatgac caagctccgg gcatccacca cgtctgagac cattcagtac 2160 aggaaggctg tgaaagccac tctggtgctg ctgcccctcc tgggcatcac ctacatgctg 2220 ttcttcgtca atcccgggga ggatgaggtc tcccgggtcg tcttcatcta cttcaactcc 2280 ttcctggaat ccttccaggg cttctttgtg tctgtgttct actgtttcct caatagtgag 2340 gtccgttctg ccatccggaa gaggtggcac cggtggcagg acaagcactc gatccgtgcc 2400 cgagtggccc gtgccatgtc catccccacc tccccaaccc gtgtcagctt tcacagcatc 2460 aagcagtcca cagcagtctg aggatccccg ggtaaaagga atgtctccct tgccagtact 2520 gctagggttt ttctttcaaa ctatggaagc ccattcaagc tgcatattac gattttgttt 2580 ttcgctttta gaaagtggtt tagatgagat aatagaaaaa ttcttgatct ccgacaacga 2640 gtacttttat tttttttgct aatcacttta ctcaatatta gctcgaaatc gtagaaacgt 2700 agacgggtgc gggataccga gtggtgtagt taagaatttt tataaaccac gtggcccaaa 2760 aatatgaacc caaaacgttt atacatgagt atactttaag aaggctatac cccttcgtgt 2820 tagatgtagt tttagctacc caacccgagt ctatgagctt gacttcagat gtagaaggca 2880 ttaaatcgtt ttgaatatta attaaaaaac gatgaaaatt aaatatttaa aagcaatcat 2940 acgctgaaaa tttagtgctg tggctaatcc ttcaacatgg aaatgccata aaagtgactt 3000 tgacaaaaaa aaaagtatat acaggtagta aactcatcta cttcattgac tttgtttaca 3060 gcatgtggaa ggaggaatat ttattgctaa atcgtagttt aacattcaat aagtaatact 3120 attgaaattc gacaagattg gccgcatgga tgaaaaagag gcattttgct ttgggagaat 3180 tagttcaaat tagaactgaa aaaaaaaact ttacgaggca aaaatgtcgg attgagatcg 3240 taaaagttcg ctcgtcgtct tttgctttgt gattgttttc atggatacat cttgctggat 3300 atttaaattt tagtactatg tataagatat tctataaatg ttttatcacc caaacctgtt 3360 agcgccttct taattctatt caatctggct tttgctctga gactacttct tggactttca 3420 ctacttgtta gttatacgga atttgtgtaa ttagaagtga aataatcctt tctattagta 3480 atgcgagctc gaattcgagt ctaactcctt aaccactcgg acatagtgac ttatctgaca 3540 ctcattgtaa attaatatat atataggcat tttgtttagt taaaggtact taagtaatta 3600 gtataaacga accaatttta taatcaggaa gttaagtgaa tggtagcaca tgtcgtaaaa 3660 attgtgaatt tttattgaat aatattttaa atacaagcct ttctagacta ggtatactca 3720 taaacatata tgagcaaaag gatagaggag attacattgc atcttctaca aatttattta 3780 ttgcccttta ctgaaaaatt aaataatgag tactaaatga taaaaagcgc tcagtacaag 3840 aaagattgca aaaatattgc attcttcatg aattaaagtt gcatataagg catattgaaa 3900 gtaatagtac taaaacagca gttagcgaaa attaatagaa ttatattcgc aagacaattg 3960 tgacattaaa ttaaaaaatt gtagaatttt tactatcctc tttaacgcca tgagccttta 4020 taaaaaggtt aaattagttt taacattctt tttttgagta agagtttaca atttatcaaa 4080 acctgtgtta ttatattcat tagtttcaat ttattagcat ctagagaaaa atcaattggc 4140 agttacattg ttggaattta tgaagaaaaa gaatctacaa cggagaataa gttgctgatc 4200 gctttcccta aaattgtata ttttgctgag cttattttga catttcgttg aagttttctc 4260 taattcgcat tcattttaag taaaacaatg agaaataaaa ttacaaaaaa tacaaattaa 4320 aatacaattt ttagctataa ttatagacga tgcccttgta tcccattctg tctcgcttgc 4380 ccctactttt tatcttttat ataccataat gaacgctgcc gctactaacc ataccccgat 4440 tttacatttc ggactcccaa ggacgtacaa aatagaaaac tatagaaaaa aataatcaga 4500 aaatagcatg tcatctcttt gtaaaacgcg tttgcaagaa gaaaggaaac aatggagaag 4560 agatcatcca tttgtatgta aattttagta aacttgaaga aatcactaac aacttctctt 4620 acttagggat tctatgcaaa accttgtaaa tcatctgatg gaggactcga tttaatgaat 4680 tggaaggttg gaattcactg gccgtcgttt tacaacgtcg tgactgggaa aaccctggcg 4740 ttacccaact taatcgcctt gcagcacatc cccctttcgc cagctggcgt aatagcgaag 4800 aggcccgcac cgatcgccct tcccaacagt tgcgcagcct gaatggcgaa tggcgcctga 4860 tgcggtattt tctccttacg catctgtgcg gtatttcaca ccgcatacgt caaagcaacc 4920 atagtacgcg ccctgtagcg gcgcattaag cgcggcgggt gtggtggtta cgcgcagcgt 4980 gaccgctaca cttgccagcg ccttagcgcc cgctcctttc gctttcttcc cttcctttct 5040 cgccacgttc gccggctttc cccgtcaagc tctaaatcgg gggctccctt tagggttccg 5100 atttagtgct ttacggcacc tcgaccccaa aaaacttgat ttgggtgatg gttcacgtag 5160 tgggacatcg ccctgataga cggtttttcg ccctttgacg ttggagtcca cgttctttaa 5220 tagtggactc ttgttccaaa ctggaacaac actcaactct atctcgggct attcttttga 5280 tttataaggg attttgccga tttcggtcta ttggttaaaa aatgagctga tttaacaaaa 5340 atttaacgcg aattttaaca aaatattaac gtttacaatt ttatggtgca ctctcagtac 5400 aatctgctct gatgccgcat agttaagcca gccccgacac ccgccaacac ccgctgacgc 5460 gccctgacgg gcttgtctgc tcccggcatc cgcttacaga caagctgtga ccgtctccgg 5520 gagctgcatg tgtcagaggt tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct 5580 cgtgatacgc ctatttttat aggttaatgt catgataata atggtttctt agacgtcagg 5640 tggcactttt cggggaaatg tgcgcggaac ccctatttgt ttatttttct aaatacattc 5700 aaatatgtat ccgctcatga gacaataacc ctgataaatg cttcaataat attgaaaaag 5760 gaagagtatg agtattcaac atttccgtgt cgcccttatt cccttttttg cggcattttg 5820 ccttcctgtt tttgctcacc cagaaacgct ggtgaaagta aaagatgctg aagatcagtt 5880 gggtgcacga gtgggttaca tcgaactgga tctcaacagc ggtaagatcc ttgagagttt 5940 tcgccccgaa gaacgttttc caatgatgag cacttttaaa gttctgctat gtggcgcggt 6000 attatcccgt attgacgccg ggcaagagca actcggtcgc cgcatacact attctcagaa 6060 tgacttggtt gagtactcac cagtcacaga aaagcatctt acggatggca tgacagtaag 6120 agaattatgc agtgctgcca taaccatgag tgataacact gcggccaact tacttctgac 6180 aacgatcgga ggaccgaagg agctaaccgc ttttttgcac aacatggggg atcatgtaac 6240 tcgccttgat cgttgggaac cggagctgaa tgaagccata ccaaacgacg agcgtgacac 6300 cacgatgcct gtagcaatgg caacaacgtt gcgcaaacta ttaactggcg aactacttac 6360 tctagcttcc cggcaacaat taatagactg gatggaggcg gataaagttg caggaccact 6420 tctgcgctcg gcccttccgg ctggctggtt tattgctgat aaatctggag ccggtgagcg 6480 tgggtctcgc ggtatcattg cagcactggg gccagatggt aagccctccc gtatcgtagt 6540 tatctacacg acggggagtc aggcaactat ggatgaacga aatagacaga tcgctgagat 6600 aggtgcctca ctgattaagc attggtaact gtcagaccaa gtttactcat atatacttta 6660 gattgattta aaacttcatt tttaatttaa aaggatctag gtgaagatcc tttttgataa 6720 tctcatgacc aaaatccctt aacgtgagtt ttcgttccac tgagcgtcag accccgtaga 6780 aaagatcaaa ggatcttctt gagatccttt ttttctgcgc gtaatctgct gcttgcaaac 6840 aaaaaaacca ccgctaccag cggtggtttg tttgccggat caagagctac caactctttt 6900 tccgaaggta actggcttca gcagagcgca gataccaaat actgtccttc tagtgtagcc 6960 gtagttaggc caccacttca agaactctgt agcaccgcct acatacctcg ctctgctaat 7020 cctgttacca gtggctgctg ccagtggcga taagtcgtgt cttaccgggt tggactcaag 7080 acgatagtta ccggataagg cgcagcggtc gggctgaacg gggggttcgt gcacacagcc 7140 cagcttggag cgaacgacct acaccgaact gagataccta cagcgtgagc tatgagaaag 7200 cgccacgctt cccgaaggga gaaaggcgga caggtatccg gtaagcggca gggtcggaac 7260 aggagagcgc acgagggagc ttccaggggg aaacgcctgg tatctttata gtcctgtcgg 7320 gtttcgccac ctctgacttg agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct 7380 atggaaaaac gccagcaacg cggccttttt acggttcctg gccttttgct ggccttttgc 7440 tcacatgttc tttcctgcgt tatcccctga ttctgtggat aaccgtatta ccgcctttga 7500 gtgagctgat accgctcgcc gcagccgaac gaccgagcgc agcgagtcag tgagcgagga 7560 agcggaagag cgcccaatac gcaaaccgcc tctccccgcg cgttggccga ttcattaatg 7620 cagctggcac gacaggtttc ccgactggaa agcgggcagt gagcgcaacg caattaatgt 7680 gagttagctc actcattagg caccccaggc tttacacttt atgcttccgg ctcgtatgtt 7740 gtgtggaatt gtgagcggat aacaatttca cacaggaaac agctatgacc atgattacgc 7800 caagcttgtc gatcgactac gtcgttaagg ccgtttctga cagagtaaaa ttcttgaggg 7860 aactttcacc attatgggaa atggttcaag aaggtattga cttaaactcc atcaaatggt 7920 caggtcattg agtgtttttt atttgttgta tttttttttt tttagagaaa atcctccaat 7980 atcaaattag gaatcgtagt ttcatgattt tctgttacac ctaacttttt gtgtggtgcc 8040 ctcctccttg tcaatattaa tgttaaagtg caattctttt tccttatcac gttgagccat 8100 tagtatcaat ttgcttacct gtattccttt actatcctcc tttttctcct tcttgataaa 8160 tgtatgtaga ttgcgtatat agtttcgtct accctatgaa catattccat tttgtaattt 8220 cgtgtcgttt ctattatgaa tttcatttat aaagtttatg tacaaatatc ataaaaaaag 8280 agaatctttt taagcaagga ttttcttaac ttcttcggcg acagcatcac cgacttcggt 8340 ggtactgttg gaaccaccta aatcaccagt tctgatacct gcatccaaaa cctttttaac 8400 tgcatcttca atggccttac cttcttcagg caagttcaat gacaatttca acatcattgc 8460 agcagacaag atagtggcga tagggtcaac cttattcttt ggcaaatctg gagcagaacc 8520 gtggcatggt tcgtacaaac caaatgcggt gttcttgtct ggcaaagagg ccaaggacgc 8580 agatggcaac aaacccaagg aacctgggat aacggaggct tcatcggaga tgatgtcacc 8640 aaacatgttg ctggtgatta taataccatt taggtgggtt gggttcttaa ctaggatcat 8700 ggcggcagaa tcaatcaatt gatgttgaac cttcaatgta gggaattcgt tcttgatggt 8760 ttcctccaca gtttttctcc ataatcttga agaggccaaa acattagctt tatccaagga 8820 ccaaataggc aatggtggct catgttgtag ggccatgaaa gcggccattc ttgtgattct 8880 ttgcacttct ggaacggtgt attgttcact atcccaagcg acaccatcac catcgtcttc 8940 ctttctctta ccaaagtaaa tacctcccac taattctctg acaacaacga agtcagtacc 9000 tttagcaaat tgtggcttga ttggagataa gtctaaaaga gagtcggatg caaagttaca 9060 tggtcttaag ttggcgtaca attgaagttc tttacggatt tttagtaaac cttgttcagg 9120 tctaacacta ccggtacccc atttaggacc acccacagca cctaacaaaa cggcatcaac 9180 cttcttggag gcttccagcg cctcatctgg aagtgggaca cctgtagcat cgatagcagc 9240 accaccaatt aaatgatttt cgaaatcgaa cttgacattg gaacgaacat cagaaatagc 9300 tttaagaacc ttaatggctt cggctgtgat ttcttgacca acgtggtcac ctggcaaaac 9360 gacgatcttc ttaggggcag acataggggc agacattaga atggtatatc cttgaaatat 9420 atatatatat tgctgaaatg taaaaggtaa gaaaagttag aaagtaagac gattgctaac 9480 cacctattgg aaaaaacaat aggtccttaa ataatattgt caacttcaag tattgtgatg 9540 caagcattta gtcatgaacg cttctctatt ctatatgaaa agccggttcc ggcctctcac 9600 ctttcctttt tctcccaatt tttcagttga aaaaggtata tgcgtcaggc gacctctgaa 9660 attaacaaaa aatttccagt catcgaattt gattctgtgc gatagcgccc ctgtgtgttc 9720 tcgttatgtt gaggaaaaaa ataatggttg ctaagagatt cgaactcttg catcttacga 9780 tacctgagta ttcccacagt taactgcggt caagatattt cttgaatcag gcgccttaga 9840 ccgctcggcc aaacaaccaa ttacttgttg agaaatagag tataattatc ctataaatat 9900 aacgtttttg aacacacatg aacaaggaag tacaggacaa ttgattttga agagaatgtg 9960 gattttgatg taattgttgg gattccattt ttaataaggc aataatatta ggtatgtaga 10020 tatactagaa gttctcctcg ac 10042 31 5446 DNA Artificial Sequence Description of Artificial SequencepSP72 sxa2 31 gaactcgagc agctgaagct tgcatgcctg caggtcgact ctagaggatc ctataatggt 60 cacttctctt gatccttacc gtcatattgg ctgtactggt agtccttctg gttcacataa 120 tttaatttgg atgactgtct acaaagacaa actccgccgt tgtcctgaat gcggatctgt 180 atacaaatta aaattcatgg gagatcccaa cgctgaacac agtcattagt atgcttacca 240 atcttccata tccccttgct attttcatac tcttttataa caatttagct cttttatttg 300 aactttgaaa aatcaatgaa aactgtatgc tgatctattt cttattttgg gaacgattcg 360 attattcgta aacgtatatc ttataaataa ttctatctat tcaaccatat cttagttgtt 420 aattcaagca ccattctttt tttcttgtag tcatgcggaa atatgatcta attatcgttc 480 cgagtttcat aaacgatagt tatttattga tataccacat cagtggtaag tcaataaact 540 tgtattttaa attttccgat attaaacttt taataaagca acgtattttc aagccattct 600 ctaaccattt gagggtcttt agatggagcc atgtgcccaa ccgagttgga caatgtaaag 660 gctaagttac gttcatccaa agtaaatcca ttagttgtct ctaaacttcc aggagattga 720 gtgaatccct gccatccatt ccatgttgta ttttgcaatg ccaaaagagt tccggtccaa 780 agaatttgaa gatccaatgc cccagctaag aaagacactt tgtatttttc tgttaagcgt 840 ggaattattt caacgagaac tgattctacg ttgttactca caatcttttt gtataagtca 900 aaattgcatc cgtctgcgaa gacaccctcg ccgcttgtca acgcagttga agctttcgta 960 gcatgtaaag aagagcgaac atcttctctg ttcaggtatg tgattaaggg atcattatac 1020 tcattgaaac tacaatcaag gctaacatca taagttatga cacagctgtt ctctagataa 1080 agtaaaaagt tagaaaggga atacaagtca catccagaaa ttgaattaaa tacattccca 1140 atgtcttcac catcgagagc ttcccgcttc cttagagaag tagatgtgct aaagttgtac 1200 tctggtctcc atattgggta ttgttcagta ggaaacgtca atctgttcaa aactgaatca 1260 tattgacatt ctttattgcg ctttttaaac tcttcagata ttgtggaaga agtattatta 1320 aaatagtatc caagtttgct gatatgttcc acccaaatag aagcagtaat ttgttcttga 1380 gtttcgtagt ccgccgtaag tccactgacg attcccacgc ccataaaatt tatattcaaa 1440 gatggttcag aaagtaatgc ctctgcaaaa ttggcgctcc atatgctacc gtagctttcg 1500 ccgacaaggt agagcttttt cttcattaag tggggaaact tttggtaaaa tgatttcagg 1560 gcatttacaa aatctgaaga agcttcttca atcgtagttg tatatgccgc ctgtccttgg 1620 gagtaccccg taccaaatgg ctgatctaac cataacatgt tggcgaagtt tgtccaagat 1680 tcaggattaa gggatggtga ggggctactt tgggatattt caatgggtcc attttcactg 1740 aaaaagccta atgttccagc gcatccaggt cctccttgaa gccatactat gaaagtttcc 1800 gaatcaacaa cagcaggggc atatgtataa aaaagtgatt tatcggaatt cgcttccaag 1860 tatccagaat ataattccgg taatgatccc ttaaattcag gaagtgattt aattcgatcc 1920 gaagaaggag cagcgtgttg tctaggttga acagtctgat tcgaggaagc agaactcgtg 1980 ttggcttgtt gtatactgca tttccaatgt acagtgtaag ttggtaaagc gtgaatgata 2040 gtcaattcaa ttattataat agcaaaaaga gatttcaaaa ataaagacag cattgaaaag 2100 agagacaatg attttgagcc tatgagaaaa gcgttagcgc taaattaaat ataatattta 2160 tcatatttgg aatatattga tgtattcagt ttcgaaacaa ttcttgaaaa gtgactataa 2220 ttttatacgc ttcagtattt cgaaaaatgt gaaaatgcta tgacttactc gaataaagaa 2280 accggattgt ttatctgatt cattttcata agagttgcat gctatatatg ataatacata 2340 ataatgggga ttcacttgaa tagattagca ttgtccattg tttacaatca acaacaatag 2400 agatgggcaa caaagacacc cagcgaagta ccatttacta gtaataggct aattcattta 2460 agtttttcaa cacaatagtc cttaatgaaa aaaccaattg tttcgtttac gcggcaagtg 2520 aatagggtag gactagaata acctatggtc tgtttaaatc gaaaaccagc ttatttcagc 2580 agcgatcatt tatgcattct taatgcttaa caatgcgagg cgactcaaag aatggagaaa 2640 attgattgtt ttatgaccgg tggaatcgaa taatttgact tttgctatga aggctttagc 2700 ccttagaaat aaagaaataa aataaaaaat agatagataa caagaaaaaa tcaatgtcaa 2760 ataatttagt tgtattaatg aaaaataaga aataactaaa ttaaaaaaat ataaatacat 2820 ttatatatta gtaacactat ttctttttaa agaccttgaa atattatgtg agaaacttaa 2880 aaactaataa aaataaataa atataaaaat ataaagaaaa agaaaaaaat taaaaattat 2940 atatacaata tactaataaa catagtgatt aattaaatga aaaaaataaa tgtttcttaa 3000 agcatggtta aaacacacaa tggcggattt ctagccatgg taccgagctc gaattcatcg 3060 atgatatcag atctgccggt ctccctatag tgagtcgtat taatttcgat aagccaggtt 3120 aacctgcatt aatgaatcgg ccaacgcgcg gggagaggcg gtttgcgtat tgggcgctct 3180 tccgcttcct cgctcactga ctcgctgcgc tcggtcgttc ggctgcggcg agcggtatca 3240 gctcactcaa aggcggtaat acggttatcc acagaatcag gggataacgc aggaaagaac 3300 atgtgagcaa aaggccagca aaaggccagg aaccgtaaaa aggccgcgtt gctggcgttt 3360 ttccataggc tccgcccccc tgacgagcat cacaaaaatc gacgctcaag tcagaggtgg 3420 cgaaacccga caggactata aagataccag gcgtttcccc ctggaagctc cctcgtgcgc 3480 tctcctgttc cgaccctgcc gcttaccgga tacctgtccg cctttctccc ttcgggaagc 3540 gtggcgcttt ctcatagctc acgctgtagg tatctcagtt cggtgtaggt cgttcgctcc 3600 aagctgggct gtgtgcacga accccccgtt cagcccgacc gctgcgcctt atccggtaac 3660 tatcgtcttg agtccaaccc ggtaagacac gacttatcgc cactggcagc agccactggt 3720 aacaggatta gcagagcgag gtatgtaggc ggtgctacag agttcttgaa gtggtggcct 3780 aactacggct acactagaag aacagtattt ggtatctgcg ctctgctgaa gccagttacc 3840 ttcggaaaaa gagttggtag ctcttgatcc ggcaaacaaa ccaccgctgg tagcggtggt 3900 ttttttgttt gcaagcagca gattacgcgc agaaaaaaag gatctcaaga agatcctttg 3960 atcttttcta cggggtctga cgctcagtgg aacgaaaact cacgttaagg gattttggtc 4020 atgagattat caaaaaggat cttcacctag atccttttaa attaaaaatg aagttttaaa 4080 tcaatctaaa gtatatatga gtaaacttgg tctgacagtt accaatgctt aatcagtgag 4140 gcacctatct cagcgatctg tctatttcgt tcatccatag ttgcctgact ccccgtcgtg 4200 tagataacta cgatacggga gggcttacca tctggcccca gtgctgcaat gataccgcga 4260 gacccacgct caccggctcc agatttatca gcaataaacc agccagccgg aagggccgag 4320 cgcagaagtg gtcctgcaac tttatccgcc tccatccagt ctattaattg ttgccgggaa 4380 gctagagtaa gtagttcgcc agttaatagt ttgcgcaacg ttgttgccat tgctacaggc 4440 atcgtggtgt cacgctcgtc gtttggtatg gcttcattca gctccggttc ccaacgatca 4500 aggcgagtta catgatcccc catgttgtgc aaaaaagcgg ttagctcctt cggtcctccg 4560 atcgttgtca gaagtaagtt ggccgcagtg ttatcactca tggttatggc agcactgcat 4620 aattctctta ctgtcatgcc atccgtaaga tgcttttctg tgactggtga gtactcaacc 4680 aagtcattct gagaatagtg tatgcggcga ccgagttgct cttgcccggc gtcaatacgg 4740 gataataccg cgccacatag cagaacttta aaagtgctca tcattggaaa acgttcttcg 4800 gggcgaaaac tctcaaggat cttaccgctg ttgagatcca gttcgatgta acccactcgt 4860 gcacccaact gatcttcagc atcttttact ttcaccagcg tttctgggtg agcaaaaaca 4920 ggaaggcaaa atgccgcaaa aaagggaata agggcgacac ggaaatgttg aatactcata 4980 ctcttccttt ttcaatatta ttgaagcatt tatcagggtt attgtctcat gagcggatac 5040 atatttgaat gtatttagaa aaataaacaa ataggggttc cgcgcacatt tccccgaaaa 5100 gtgccacctg acgtctaaga aaccattatt atcatgacat taacctataa aaataggcgt 5160 atcacgaggc cctttcgtct cgcgcgtttc ggtgatgacg gtgaaaacct ctgacacatg 5220 cagctcccgg agacggtcac agcttgtctg taagcggatg ccgggagcag acaagcccgt 5280 cagggcgcgt cagcgggtgt tggcgggtgt cggggctggc ttaactatgc ggcatcagag 5340 cagattgtac tgagagtgca ccatatggac atattgtcgt tagaacgcgg ctacaattaa 5400 tacataacct tatgtatcat acacatacga tttaggtgac actata 5446 32 5704 DNA Artificial Sequence Description of Artificial SequencepSP72 sxa2::ura4+ 32 gaactcgagc agctgaagct tgcatgcctg caggtcgact ctagaggatc ctataatggt 60 cacttctctt gatccttacc gtcatattgg ctgtactggt agtccttctg gttcacataa 120 tttaatttgg atgactgtct acaaagacaa actccgccgt tgtcctgaat gcggatctgt 180 atacaaatta aaattcatgg gagatcccaa cgctgaacac agtcattagt atgcttacca 240 atcttccata tccccttgct attttcatac tcttttataa caatttagct cttttatttg 300 aactttgaaa aatcaatgaa aactgtatgc tgatctattt cttattttgg gaacgattcg 360 attattcgta aacgtatatc ttataaataa ttctatctat tcaaccatat cttagttgtt 420 aattcaagca ccattctttt tttcttgtag tcatgcggaa atatgatcta attatcgttc 480 cgagtttcat aaacgatagt tatttattga tataccacat cagtggtaag tcaataaact 540 tgtattttaa attttccgat attaaacttt taggatccac catgtagtga tattgacgaa 600 actttttgac atctaattta ttctgttcca acaccaatgt ttataaccaa gttttatctt 660 gtttgtctac atggtatttt acattcatct acatacatct ttcattggct ttgtacatag 720 ttatcattac aagtctaaaa aaattcactc ttttcttatt caatgtcaat ccaagagaaa 780 agattgtggt aatgttgtag gagcatgttt aataaattac tatagcaaat tactttttat 840 tcccaaggtg tttatctata atagttaata ttttagtcgc tacataaaat tttaccaaag 900 agtacttgta tactaattct aaatgccttc tgacataaaa cgcctaggaa aacaaacgca 960 aacaaggcat cgactttttc aataaccaac caaaaaaatt ttacattagt ctttttttaa 1020 tgctgagaaa gtctttgctg atatgccttc caaccagctt ctctatatct cttggcttcg 1080 acaacaggat tacgaccagc tccatagact ccacgaccaa caatgatgat atcgctaccg 1140 cagtttacaa tcacttcttc aggagtacga tattgctgtc ccagcccgtc tcctttaaca 1200 tccaagccga taccagggga catagttatg tagtcgcttt gaaggttagg aaatcgacga 1260 ccagctataa agccaaagca aaaatcggta tgcttctcaa accattctaa ggttttctct 1320 gtgtaggaac cagtagccaa agagcctttg gaagacattt cagccaaaag caagagacca 1380 cgtcccaaag gtaaaccaac ttctttgagg ccttgtataa taccctcgcc tggcactgta 1440 tggcaatttg tgatatgagc ccaagaagca attttgtaca caccagatgc atattgtagc 1500 ttgacggtat ttccaatgtc tgcgaatttg cgatcctcaa agataagaaa acgatgcttt 1560 ttacctaagg ccaccagttt ttctaccata tcctggtcga aatcctcgac aacgtcaata 1620 tgtgtcttga taacacagac atagggtcca attttatcta ccaattctaa gatttcggat 1680 ttcttcgtca aatcgaccgc gactgacaag ttgctttgct tttcttccat caaagccaac 1740 aattccttgg caatgggatt tttcatcccc tcagctctag ctgaatagct ttgaaatact 1800 ctagcatcca taactttgct tttaaacctt taatttcgat ccaagcaaaa aagaggttct 1860 tggtaggaca atacggtaag aaaacacgac atgtgcagag atgccgacga agcatagtta 1920 aactgggatg gtaaaatcaa ttaagaattt ataaagacaa aattgtataa gtctctaaaa 1980 catcttaatt atacctcaca gaactatcta aaatatattc acaaagtgca aacattatca 2040 tgaaaaagaa ccattttaat ttaaagcaag ggcattaagg cttatttaca gaatttctta 2100 cttttgtaaa gattataagg ctgattatct ttttcaccat gccaaaaatt acacaagata 2160 gaatggatgt ttgaaattaa acgtgagtat acaaacaaat acactaggta aatcgaaaca 2220 tttttttctc cattaagtaa caaattccta tttagagaaa gaatgctgag tagattaaat 2280 aatctataca aactttttta acacaaatgc atacatatag ccagtgggat ttgtagctac 2340 atggtggatc ctgaaaagag agacaatgat tttgagccta tgagaaaagc gttagcgcta 2400 aattaaatat aatatttatc atatttggaa tatattgatg tattcagttt cgaaacaatt 2460 cttgaaaagt gactataatt ttatacgctt cagtatttcg aaaaatgtga aaatgctatg 2520 acttactcga ataaagaaac cggattgttt atctgattca ttttcataag agttgcatgc 2580 tatatatgat aatacataat aatggggatt cacttgaata gattagcatt gtccattgtt 2640 tacaatcaac aacaatagag atgggcaaca aagacaccca gcgaagtacc atttactagt 2700 aataggctaa ttcatttaag tttttcaaca caatagtcct taatgaaaaa accaattgtt 2760 tcgtttacgc ggcaagtgaa tagggtagga ctagaataac ctatggtctg tttaaatcga 2820 aaaccagctt atttcagcag cgatcattta tgcattctta atgcttaaca atgcgaggcg 2880 actcaaagaa tggagaaaat tgattgtttt atgaccggtg gaatcgaata atttgacttt 2940 tgctatgaag gctttagccc ttagaaataa agaaataaaa taaaaaatag atagataaca 3000 agaaaaaatc aatgtcaaat aatttagttg tattaatgaa aaataagaaa taactaaatt 3060 aaaaaaatat aaatacattt atatattagt aacactattt ctttttaaag accttgaaat 3120 attatgtgag aaacttaaaa actaataaaa ataaataaat ataaaaatat aaagaaaaag 3180 aaaaaaatta aaaattatat atacaatata ctaataaaca tagtgattaa ttaaatgaaa 3240 aaaataaatg tttcttaaag catggttaaa acacacaatg gcggatttct agccatggta 3300 ccgagctcga attcatcgat gatatcagat ctgccggtct ccctatagtg agtcgtatta 3360 atttcgataa gccaggttaa cctgcattaa tgaatcggcc aacgcgcggg gagaggcggt 3420 ttgcgtattg ggcgctcttc cgcttcctcg ctcactgact cgctgcgctc ggtcgttcgg 3480 ctgcggcgag cggtatcagc tcactcaaag gcggtaatac ggttatccac agaatcaggg 3540 gataacgcag gaaagaacat gtgagcaaaa ggccagcaaa aggccaggaa ccgtaaaaag 3600 gccgcgttgc tggcgttttt ccataggctc cgcccccctg acgagcatca caaaaatcga 3660 cgctcaagtc agaggtggcg aaacccgaca ggactataaa gataccaggc gtttccccct 3720 ggaagctccc tcgtgcgctc tcctgttccg accctgccgc ttaccggata cctgtccgcc 3780 tttctccctt cgggaagcgt ggcgctttct catagctcac gctgtaggta tctcagttcg 3840 gtgtaggtcg ttcgctccaa gctgggctgt gtgcacgaac cccccgttca gcccgaccgc 3900 tgcgccttat ccggtaacta tcgtcttgag tccaacccgg taagacacga cttatcgcca 3960 ctggcagcag ccactggtaa caggattagc agagcgaggt atgtaggcgg tgctacagag 4020 ttcttgaagt ggtggcctaa ctacggctac actagaagaa cagtatttgg tatctgcgct 4080 ctgctgaagc cagttacctt cggaaaaaga gttggtagct cttgatccgg caaacaaacc 4140 accgctggta gcggtggttt ttttgtttgc aagcagcaga ttacgcgcag aaaaaaagga 4200 tctcaagaag atcctttgat cttttctacg gggtctgacg ctcagtggaa cgaaaactca 4260 cgttaaggga ttttggtcat gagattatca aaaaggatct tcacctagat ccttttaaat 4320 taaaaatgaa gttttaaatc aatctaaagt atatatgagt aaacttggtc tgacagttac 4380 caatgcttaa tcagtgaggc acctatctca gcgatctgtc tatttcgttc atccatagtt 4440 gcctgactcc ccgtcgtgta gataactacg atacgggagg gcttaccatc tggccccagt 4500 gctgcaatga taccgcgaga cccacgctca ccggctccag atttatcagc aataaaccag 4560 ccagccggaa gggccgagcg cagaagtggt cctgcaactt tatccgcctc catccagtct 4620 attaattgtt gccgggaagc tagagtaagt agttcgccag ttaatagttt gcgcaacgtt 4680 gttgccattg ctacaggcat cgtggtgtca cgctcgtcgt ttggtatggc ttcattcagc 4740 tccggttccc aacgatcaag gcgagttaca tgatccccca tgttgtgcaa aaaagcggtt 4800 agctccttcg gtcctccgat cgttgtcaga agtaagttgg ccgcagtgtt atcactcatg 4860 gttatggcag cactgcataa ttctcttact gtcatgccat ccgtaagatg cttttctgtg 4920 actggtgagt actcaaccaa gtcattctga gaatagtgta tgcggcgacc gagttgctct 4980 tgcccggcgt caatacggga taataccgcg ccacatagca gaactttaaa agtgctcatc 5040 attggaaaac gttcttcggg gcgaaaactc tcaaggatct taccgctgtt gagatccagt 5100 tcgatgtaac ccactcgtgc acccaactga tcttcagcat cttttacttt caccagcgtt 5160 tctgggtgag caaaaacagg aaggcaaaat gccgcaaaaa agggaataag ggcgacacgg 5220 aaatgttgaa tactcatact cttccttttt caatattatt gaagcattta tcagggttat 5280 tgtctcatga gcggatacat atttgaatgt atttagaaaa ataaacaaat aggggttccg 5340 cgcacatttc cccgaaaagt gccacctgac gtctaagaaa ccattattat catgacatta 5400 acctataaaa ataggcgtat cacgaggccc tttcgtctcg cgcgtttcgg tgatgacggt 5460 gaaaacctct gacacatgca gctcccggag acggtcacag cttgtctgta agcggatgcc 5520 gggagcagac aagcccgtca gggcgcgtca gcgggtgttg gcgggtgtcg gggctggctt 5580 aactatgcgg catcagagca gattgtactg agagtgcacc atatggacat attgtcgtta 5640 gaacgcggct acaattaata cataacctta tgtatcatac acatacgatt taggtgacac 5700 tata 5704 33 6952 DNA Artificial Sequence Description of Artificial SequencepSP72 sxa>lacZ 33 tatagtgtca cctaaatcgt atgtgtatga tacataaggt tatgtattaa ttgtagccgc 60 gttctaacga caatatgtcc atatggtgca ctctcagtac aatctgctct gatgccgcat 120 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 180 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 240 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 300 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 360 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 420 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 480 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 540 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 600 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 660 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 720 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 780 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 840 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 900 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 960 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 1020 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 1080 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 1140 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 1200 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 1260 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 1320 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 1380 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 1440 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 1500 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 1560 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 1620 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca 1680 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 1740 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 1800 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 1860 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 1920 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 1980 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 2040 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 2100 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 2160 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 2220 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac 2280 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg caggttaacc tggcttatcg 2340 aaattaatac gactcactat agggagaccg gcagatctga tatcatcgat gaattcgagc 2400 tcggtaccat ggctagaaat ccgccattgt gtgttttaac catgctttaa gaaacattta 2460 tttttttcat ttaattaatc actatgttta ttagtatatt gtatatataa tttttaattt 2520 ttttcttttt ctttatattt ttatatttat ttatttttat tagtttttaa gtttctcaca 2580 taatatttca aggtctttaa aaagaaatag tgttactaat atataaatgt atttatattt 2640 ttttaattta gttatttctt atttttcatt aatacaacta aattatttga cattgatttt 2700 ttcttgttat ctatctattt tttattttat ttctttattt ctaagggcta aagccttcat 2760 agcaaaagtc aaattattcg attccaccgg tcataaaaca atcaattttc tccattcttt 2820 gagtcgcctc gcattgttaa gcattaagaa tgcataaatg atcgctgctg aaataagctg 2880 gttttcgatt taaacagacc ataggttatt ctagtcctac cctattcact tgccgcgtaa 2940 acgaaacaat tggttttttc attaaggact attgtgttga aaaacttaaa tgaattagcc 3000 tattactagt aaatggtact tcgctgggtg tctttgttgc ccatctctat tgttgttgat 3060 tgtaaacaat ggacaatgct aatctattca agtgaatccc cattattatg tattatcata 3120 tatagcatgc aactcttatg aaaatgaatc agataaacaa tccggtttct ttattcgagt 3180 aagtcatagc attttcacat ttttcgaaat actgaagcgt ataaaattat agtcactttt 3240 caagaattgt ttcgaaactg aatacatcaa tatattccaa atatgataaa tattatattt 3300 aatttagcgc taacgctttt ctcataggct caaaatcatt gtctctcttt tcaatgcagc 3360 tggcacgaca ggtttcccga cttaatcgcc ttgcagcaca tccccctttc gccagctggc 3420 gtaatagcga agaggcccgc accgatcgcc cttcccaaca gttgcgcagc ctgaatggcg 3480 aatggcgctt tgcctggttt ccggcaccag aagcggtgcc ggaaagctgg ctggagtgcg 3540 atcttcctga ggccgatact gtcgtcgtcc cctcaaactg gcagatgcac ggttacgatg 3600 cgcccatcta caccaacgta acctatccca ttacggtcaa tccgccgttt gttcccacgg 3660 agaatccgac gggttgttac tcgctcacat ttaatgttga tgaaagctgg ctacaggaag 3720 gccagacgcg aattattttt gatggcgtta actcggcgtt tcatctgtgg tgcaacgggc 3780 gctgggtcgg ttacggccag gacagtcgtt tgccgtctga atttgacctg agcgcatttt 3840 tacgcgccgg agaaaaccgc ctcgcggtga tggtgctgcg ttggagtgac ggcagttatc 3900 tggaagatca ggatatgtgg cggatgagcg gcattttccg tgacgtctcg ttgctgcata 3960 aaccgactac acaaatcagc gatttccatg ttgccactcg ctttaatgat gatttcagcc 4020 gcgctgtact ggaggctgaa gttcagatgt gcggcgagtt gcgtgactac ctacgggtaa 4080 cagtttcttt atggcagggt gaaacgcagg tcgccagcgg caccgcgcct ttcggcggtg 4140 aaattatcga tgagcgtggt ggttatgccg atcgcgtcac actacgtctg aacgtcgaaa 4200 acccgaaact gtggagcgcc gaaatcccga atctctatcg tgcggtggtt gaactgcaca 4260 ccgccgacgg cacgctgatt gaagcagaag cctgcgatgt cggtttccgc gaggtgcgga 4320 ttgaaaatgg tctgctgctg ctgaacggca agccgttgct gattcgaggc gttaaccgtc 4380 acgagcatca tcctctgcat ggtcaggtca tggatgagca gacgatggtg caggatatcc 4440 tgctgatgaa gcagaacaac tttaacgccg tgcgctgttc gcattatccg aaccatccgc 4500 tgtggtacac gctgtgcgac cgctacggcc tgtatgtggt ggatgaagcc aatattgaaa 4560 cccacggcat ggtgccaatg aatcgtctga ccgatgatcc gcgctggcta ccggcgatga 4620 gcgaacgcgt aacgcgaatg gtgcagcgcg atcgtaatca cccgagtgtg atcatctggt 4680 cgctggggaa tgaatcaggc cacggcgcta atcacgacgc gctgtatcgc tggatcaaat 4740 ctgtcgatcc ttcccgcccg gtgcagtatg aaggcggcgg agccgacacc acggccaccg 4800 atattatttg cccgatgtac gcgcgcgtgg atgaagacca gcccttcccg gctgtgccga 4860 aatggtccat caaaaaatgg ctttcgctac ctggagagac gcgcccgctg atcctttgcg 4920 aatacgccca cgcgatgggt aacagtcttg gcggtttcgc taaatactgg caggcgtttc 4980 gtcagtatcc ccgtttacag ggcggcttcg tctgggactg ggtggatcag tcgctgatta 5040 aatatgatga aaacggcaac ccgtggtcgg cttacggcgg tgattttggc gatacgccga 5100 acgatcgcca gttctgtatg aacggtctgg tctttgccga ccgcacgccg catccagcgc 5160 tgacggaagc aaaacaccag cagcagtttt tccagttccg tttatccggg caaaccatcg 5220 aagtgaccag cgaatacctg ttccgtcata gcgataacga gctcctgcac tggatggtgg 5280 cgctggatgg taagccgctg gcaagcggtg aagtgcctct ggatgtcgct ccacaaggta 5340 aacagttgat tgaactgcct gaactaccgc agccggagag cgccgggcaa ctctggctca 5400 cagtacgcgt agtgcaaccg aacgcgaccg catggtcaga agccgggcac atcagcgcct 5460 ggcagcagtg gcgtctggcg gaaaacctca gtgtgacgct ccccgccgcg tcccacgcca 5520 tcccgcatct gaccaccagc gaaatggatt tttgcatcga gctgggtaat aagcgttggc 5580 aatttaaccg ccagtcaggc tttctttcac agatgtggat tggcgataaa aaacaactgc 5640 tgacgccgct gcgcgatcag ttcacccgtg caccgctgga taacgacatt ggcgtaagtg 5700 aagcgacccg cattgaccct aacgcctggg tcgaacgctg gaaggcggcg ggccattacc 5760 aggccgaagc agcgttgttg cagtgcacgg cagatacact tgctgatgcg gtgctgatta 5820 cgaccgctca cgcgtggcag catcagggga aaaccttatt tatcagccgg aaaacctacc 5880 ggattgatgg tagtggtcaa atggcgatta ccgttgatgt tgaagtggcg agcgatacac 5940 cgcatccggc gcggattggc ctgaactgcc agctggcgca ggtagcagag cgggtaaact 6000 ggctcggatt agggccgcaa gaaaactatc ccgaccgcct tactgccgcc tgttttgacc 6060 gctgggatct gccattgtca gacatgtata ccccgtacgt cttcccgagc gaaaacggtc 6120 tgcgctgcgg gacgcgcgaa ttgaattatg gcccacacca gtggcgcggc gacttccagt 6180 tcaacatcag ccgctacagt caacagcaac tgatggaaac cagccatcgc catctgctgc 6240 acgcggaaga aggcacatgg ctgaatatcg acggtttcca tatggggatt ggtggcgacg 6300 actcctggag cccgtcagta tcggcggaat tccagctgag cgccggtcgc taccattacc 6360 agttggtctg gtgtcaaaaa taaaagttta atatcggaaa atttaaaata caagtttatt 6420 gacttaccac tgatgtggta tatcaataaa taactatcgt ttatgaaact cggaacgata 6480 attagatcat atttccgcat gactacaaga aaaaaagaat ggtgcttgaa ttaacaacta 6540 agatatggtt gaatagatag aattatttat aagatatacg tttacgaata atcgaatcgt 6600 tcccaaaata agaaatagat cagcatacag ttttcattga tttttcaaag ttcaaataaa 6660 agagctaaat tgttataaaa gagtatgaaa atagcaaggg gatatggaag attggtaagc 6720 atactaatga ctgtgttcag cgttgggatc tcccatgaat tttaatttgt atacagatcc 6780 gcattcagga caacggcgga gtttgtcttt gtagacagtc atccaaatta aattatgtga 6840 accagaagga ctaccagtac agccaatatg acggtaagga tcaagagaag tgaccattat 6900 aggatcctct agagtcgacc tgcaggcatg caagcttcag ctgctcgagt tc 6952 34 4717 DNA Artificial Sequence Description of Artificial SequencepSP72 sxa2>ura4 34 tatagtgtca cctaaatcgt atgtgtatga tacataaggt tatgtattaa ttgtagccgc 60 gttctaacga caatatgtcc atatggtgca ctctcagtac aatctgctct gatgccgcat 120 agttaagcca gccccgacac ccgccaacac ccgctgacgc gccctgacgg gcttgtctgc 180 tcccggcatc cgcttacaga caagctgtga ccgtctccgg gagctgcatg tgtcagaggt 240 tttcaccgtc atcaccgaaa cgcgcgagac gaaagggcct cgtgatacgc ctatttttat 300 aggttaatgt catgataata atggtttctt agacgtcagg tggcactttt cggggaaatg 360 tgcgcggaac ccctatttgt ttatttttct aaatacattc aaatatgtat ccgctcatga 420 gacaataacc ctgataaatg cttcaataat attgaaaaag gaagagtatg agtattcaac 480 atttccgtgt cgcccttatt cccttttttg cggcattttg ccttcctgtt tttgctcacc 540 cagaaacgct ggtgaaagta aaagatgctg aagatcagtt gggtgcacga gtgggttaca 600 tcgaactgga tctcaacagc ggtaagatcc ttgagagttt tcgccccgaa gaacgttttc 660 caatgatgag cacttttaaa gttctgctat gtggcgcggt attatcccgt attgacgccg 720 ggcaagagca actcggtcgc cgcatacact attctcagaa tgacttggtt gagtactcac 780 cagtcacaga aaagcatctt acggatggca tgacagtaag agaattatgc agtgctgcca 840 taaccatgag tgataacact gcggccaact tacttctgac aacgatcgga ggaccgaagg 900 agctaaccgc ttttttgcac aacatggggg atcatgtaac tcgccttgat cgttgggaac 960 cggagctgaa tgaagccata ccaaacgacg agcgtgacac cacgatgcct gtagcaatgg 1020 caacaacgtt gcgcaaacta ttaactggcg aactacttac tctagcttcc cggcaacaat 1080 taatagactg gatggaggcg gataaagttg caggaccact tctgcgctcg gcccttccgg 1140 ctggctggtt tattgctgat aaatctggag ccggtgagcg tgggtctcgc ggtatcattg 1200 cagcactggg gccagatggt aagccctccc gtatcgtagt tatctacacg acggggagtc 1260 aggcaactat ggatgaacga aatagacaga tcgctgagat aggtgcctca ctgattaagc 1320 attggtaact gtcagaccaa gtttactcat atatacttta gattgattta aaacttcatt 1380 tttaatttaa aaggatctag gtgaagatcc tttttgataa tctcatgacc aaaatccctt 1440 aacgtgagtt ttcgttccac tgagcgtcag accccgtaga aaagatcaaa ggatcttctt 1500 gagatccttt ttttctgcgc gtaatctgct gcttgcaaac aaaaaaacca ccgctaccag 1560 cggtggtttg tttgccggat caagagctac caactctttt tccgaaggta actggcttca 1620 gcagagcgca gataccaaat actgttcttc tagtgtagcc gtagttaggc caccacttca 1680 agaactctgt agcaccgcct acatacctcg ctctgctaat cctgttacca gtggctgctg 1740 ccagtggcga taagtcgtgt cttaccgggt tggactcaag acgatagtta ccggataagg 1800 cgcagcggtc gggctgaacg gggggttcgt gcacacagcc cagcttggag cgaacgacct 1860 acaccgaact gagataccta cagcgtgagc tatgagaaag cgccacgctt cccgaaggga 1920 gaaaggcgga caggtatccg gtaagcggca gggtcggaac aggagagcgc acgagggagc 1980 ttccaggggg aaacgcctgg tatctttata gtcctgtcgg gtttcgccac ctctgacttg 2040 agcgtcgatt tttgtgatgc tcgtcagggg ggcggagcct atggaaaaac gccagcaacg 2100 cggccttttt acggttcctg gccttttgct ggccttttgc tcacatgttc tttcctgcgt 2160 tatcccctga ttctgtggat aaccgtatta ccgcctttga gtgagctgat accgctcgcc 2220 gcagccgaac gaccgagcgc agcgagtcag tgagcgagga agcggaagag cgcccaatac 2280 gcaaaccgcc tctccccgcg cgttggccga ttcattaatg caggttaacc tggcttatcg 2340 aaattaatac gactcactat agggagaccg gcagatctga tatcatcgat gaattcgagc 2400 tcggtaccat ggctagaaat ccgccattgt gtgttttaac catgctttaa gaaacattta 2460 tttttttcat ttaattaatc actatgttta ttagtatatt gtatatataa tttttaattt 2520 ttttcttttt ctttatattt ttatatttat ttatttttat tagtttttaa gtttctcaca 2580 taatatttca aggtctttaa aaagaaatag tgttactaat atataaatgt atttatattt 2640 ttttaattta gttatttctt atttttcatt aatacaacta aattatttga cattgatttt 2700 ttcttgttat ctatctattt tttattttat ttctttattt ctaagggcta aagccttcat 2760 agcaaaagtc aaattattcg attccaccgg tcataaaaca atcaattttc tccattcttt 2820 gagtcgcctc gcattgttaa gcattaagaa tgcataaatg atcgctgctg aaataagctg 2880 gttttcgatt taaacagacc ataggttatt ctagtcctac cctattcact tgccgcgtaa 2940 acgaaacaat tggttttttc attaaggact attgtgttga aaaacttaaa tgaattagcc 3000 tattactagt aaatggtact tcgctgggtg tctttgttgc ccatctctat tgttgttgat 3060 tgtaaacaat ggacaatgct aatctattca agtgaatccc cattattatg tattatcata 3120 tatagcatgc aactcttatg aaaatgaatc agataaacaa tccggtttct ttattcgagt 3180 aagtcatagc attttcacat ttttcgaaat actgaagcgt ataaaattat agtcactttt 3240 caagaattgt ttcgaaactg aatacatcaa tatattccaa atatgataaa tattatattt 3300 aatttagcgc taacgctttt ctcataggct caaaatcatt gtctctcttt tcaatggatg 3360 ctagagtatt tcaaagctat tcagctagag ctgaggggat gaaaaatccc attgccaagg 3420 aattgttggc tttgatggaa gaaaagcaaa gcaacttgtc agtcgcggtc gatttgacga 3480 agaaatccga aatcttagaa ttggtagata aaattggacc ctatgtctgt gttatcaaga 3540 cacatattga cgttgtcgag gatttcgacc aggatatggt agaaaaactg gtggccttag 3600 gtaaaaagca tcgttttctt atctttgagg atcgcaaatt cgcagacatt ggaaataccg 3660 tcaagctaca atatgcatct ggtgtgtaca aaattgcttc ttgggctcat atcacaaatt 3720 gccatacagt gccaggcgag ggtattatac aaggcctcaa agaagttggt ttacctttgg 3780 gacgtggtct cttgcttttg gctgaaatgt cttccaaagg ctctttggct actggttcct 3840 acacagagaa aaccttagaa tggtttgaga agcataccga tttttgcttt ggctttatag 3900 ctggtcgtcg atttcctaac cttcaaagcg actacataac tatgtcccct ggtatcggct 3960 tggatgttaa aggagacggg ctgggacagc aatatcgtac tcctgaagaa gtgattgtaa 4020 actgcggtag cgatatcatc attgttggtc gtggagtcta tggagctggt cgtaatcctg 4080 ttgtcgaagc caagagatat agagaagctg gttggaaggc atatcagcaa agactttctc 4140 agcattaaaa gtttaatatc ggaaaattta aaatacaagt ttattgactt accactgatg 4200 tggtatatca ataaataact atcgtttatg aaactcggaa cgataattag atcatatttc 4260 cgcatgacta caagaaaaaa agaatggtgc ttgaattaac aactaagata tggttgaata 4320 gatagaatta tttataagat atacgtttac gaataatcga atcgttccca aaataagaaa 4380 tagatcagca tacagttttc attgattttt caaagttcaa ataaaagagc taaattgtta 4440 taaaagagta tgaaaatagc aaggggatat ggaagattgg taagcatact aatgactgtg 4500 ttcagcgttg ggatctccca tgaattttaa tttgtataca gatccgcatt caggacaacg 4560 gcggagtttg tctttgtaga cagtcatcca aattaaatta tgtgaaccag aaggactacc 4620 agtacagcca atatgacggt aaggatcaag agaagtgacc attataggat cctctagagt 4680 cgacctgcag gcatgcaagc ttcagctgct cgagttc 4717 35 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 35 ggggggtacc atggctagaa atccgccatt gtgtg 35 36 24 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 36 cttctcgtaa aggcacattg acgg 24 37 17 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 37 tgaaaagaga gacaatg 17 38 15 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 38 taaaagttta atatc 15 39 28 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 39 atgcagctgg cacgacaggt ttcccgac 28 40 32 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 40 tttttgacac cagaccaact ggtaatggta gc 32 41 19 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 41 atggatgcta gagtatttc 19 42 19 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 42 atgctgagaa agtctttgc 19 43 32 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 43 tagattgttg gacataatcg tatcttgaac gg 32 44 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 44 gaatataatc ttgtttagat gaatttttcc ttaac 35 45 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 45 caatatgaac ttctttagat gaatttttcc ttaac 35 46 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 46 ggatgcggac tttattagat gaatttttcc ttaac 35 47 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 47 gattgcggac ttttttagat gaatttttcc ttaac 35 48 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 48 gaatgcggac tttattagat gaatttttcc ttaac 35 49 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 49 tatattggac tttgctagat gaatttttcc ttaac 35 50 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 50 gatattatgc ttcaatagat gaatttttcc ttaac 35 51 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 51 caacttatgc ttcaatagat gaatttttcc ttaac 35 52 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 52 gaatttaatc ttgtttagat gaatttttcc ttaac 35 53 35 DNA Artificial Sequence Description of Artificial Sequence PCR primer used in the construction of the yeast and the G alpha transplants 53 gaaattaatc ttctttagat gaatttttcc ttaac 35 

1. A Schizosaccharomyces pombe (Sz pombe yeast cell comprising: (i) a G-Protein Coupled Receptor (GPCR)-regulated signalling pathway which is derepressed during the mitotic phase of cell growth; (ii) a reporter system for reporting a signal mediated by the GPCR-regulated signalling pathway; and wherein: (a) the GPCR is heterologous, and/or (b) the reporter system comprises a reporter gene and a promoter, the reporter gene being operatively linked to the promoter, and the promoter being regulatable by the GPCR, at least one of the reporter gene and the promoter being heterologous.
 2. A yeast cell as claimed in claim 1, wherein the GPCR-regulated signalling pathway is derepressed during the mitotic phase of cell growth by disruption of a nutritional control pathway.
 3. A yeast cell according to claim 1 or claim 2, wherein the yeast cell is adenylate cyclase deficient.
 4. A yeast cell according to any preceding claim which comprises a mutated pat] gene, or in which the endogenous pat1 gene has been deleted.
 5. A yeast cell as claimed in any one of claims 1 to 4, wherein the reporter system comprises a reporter gene and a promoter, the reporter gene being operatively linked to the promoter, and the promoter being regulatable by the GPCR, at least one of the reporter gene and the promoter being heterologous.
 6. A yeast cell as claimed in claim 5, wherein the reporter gene is heterologous.
 7. A yeast cell according to any preceding claim, wherein the GPCR is a mammalian GPCR.
 8. A yeast cell according to any preceding claim comprising a heterologous or chimeric G-protein subunit.
 9. A yeast cell according to claim 8, wherein the chimeric G-protein subunit is a Gα-transplant.
 10. A yeast cell according to claim 9 wherein the Gα-transplant is selected from the following transplants: Gαq (SEQ ID 17) Gαs (SEQ ID 16) Gαo (SEQ ID 18) Gαi2 (SEQ ID 19) Gαi3 (SEQ ID 20) Gαz (SEQ ID 21) Gα12 (SEQ ID 22) Gα13 (SEQ ID 23) Gα14 (SEQ ID 24) and Gα16 (SEQ ID 25)
 11. A yeast cell according to claim 5, wherein the reporter system is regulated by yeast mating pheromone binding to its GPCR
 12. A yeast cell according to claim 11, wherein the yeast mating pheromone is P-factor pheromone.
 13. A yeast cell according to claim 12, wherein the reporter system-is operatively linked to an sxa2 promoter or a homologue or analogue thereof.
 14. A yeast cell according to any preceding claim wherein the reporter system is integrated into the chromosome of the yeast cell.
 15. A yeast cell according to any preceding claim, wherein the yeast cell has a stable mating type.
 16. A yeast cell according to any preceding claim wherein the yeast cell is rgs1 deficient.
 17. A yeast cell according to any preceding claim, wherein the yeast cell is pmp1 deficient.
 18. A yeast cell according to any preceding claim, wherein the yeast cell is sxa2 deficient.
 19. A yeast cell according to claim 18, wherein at least a part of the endogenous sxa2 gene has been deleted.
 20. A yeast cell according to claim 19, wherein the reporter gene replaces the deleted mra2 gene.
 21. A yeast cell according to any preceding claim wherein the reporter gene encodes orotidine-5′-phosphate decarboxylase (the product of the Sz. pombe ura4 gene), β-galactosidase (the product of the bacterial lacZ gene), β-lactamase, aequorin, green fluorescent protein or luciferase.
 22. A yeast cell which is Schizosaccharomyces pombe strain JY546 deposited as accession number NCYC2984.
 23. A yeast cell according to any preceding claim additionally comprising one or more compounds, to be assayed for their effect on GPCR-regulated expression of the reporter system, or a DNA molecule encoding one or more peptides or proteins to be assayed
 24. A yeast cell according to claim 23, comprising one or more plasmids encoding the or each peptide or protein.
 25. A yeast cell according to claim 24, wherein the DNA encoding peptide or protein is transcribed under the control of a thiamine-regulated nmt1 promoter.
 26. A yeast cell according to claim 23 or claim 24, wherein the peptide or protein is of a random sequence.
 27. A yeast cell according to any preceding claim, wherein the yeast cell expresses an orphan receptor as the GPCR and the reporter system is regulatable by the orphan receptor.
 28. An isolated nucleic acid molecule comprising an sxa2 promoter, or a homologue or analogue thereof, operatively linked to an exogenous reporter gene.
 29. An isolated nucleic acid molecule according to claim 28, wherein the reporter system encodes orotidine-5′-phosphate decarboxylase (the product of the Sz. pombe ura4 gene), β-galactosidase (the product of the bacterial lacZ gene), a β-lactamase, aequorin, green fluorescent protein or luciferase.
 30. An isolated nucleic acid molecule encoding a Gα-transplant having a nucleic acid sequence selected from: Gαq (SEQ ID 17) Gαs (SEQ ID 16) Gαo (SEQ ID 18) Gαi2 (SEQ ID 19) Gαi3 (SEQ ID 20) Gαz (SEQ ID 21) Gα12 (SEQ ID 22) Gα13 (SEQ ID 23) Gα14 (SEQ ID 24) and Gα16 (SEQ ID 25); or which differs from the one or more of the sequences due to degeneracy in the genetic code.
 31. Use of: (a) a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising: (i) a G-Protein Coupled Receptor (GPCR)-regulated signalling pathway which is derepressed during the mitotic phase of cell growth; (ii) a reporter system for reporting a signal mediated by the GPCR-regulated signalling pathway; or; (b) an isolated nucleic acid molecule according to any one of claims 28 to 30 to study GPCR-regulated activity.
 32. An assay comprising the use of a yeast cell or isolated DNA molecule according to any one of claims to
 31. 33. A method of determining the effect of a compound, on GPCR-regulated activity comprising the steps of (i) providing a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising: (a) a G-Protein Coupled Receptor (GPCR)regulated signalling pathway which is derepressed during the mitotic phase of cell growth; (b) a reporter system for reporting a signal mediated by the GPCR-regulated signalling pathway; (ii) introducing the compound, to the yeast cell; and (iii) noting the output of the reporter system.
 34. Use of a yeast cell according to claim 27 to identify compounds, which affect the ability of the orphan GPCR to regulate the reporter system.
 35. Use of a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising: (i) a G-Protein Coupled Receptor (GPCR)-regulated signalling pathway which is derepressed during the mitotic phase of cell growth; (ii) a reporter system for reporting a signal mediated by the GPCR-regulated signalling pathway; to identify a regulator or a mutant of a component of a GPCR-regulated pathway.
 36. A method of identifying a reagent that modulates GPCR-regulated signalling pathways comprising: (i) providing a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising: (a) a G-Protein Coupled Receptor (GPCR)-regulated si pathway which is derepressed during the mitotic phase of cell growth; (b) a reporter system for reporting a signal mediated by the GPCR-regulated signaling pathway, (ii) producing a random peptide within the yeast cell; and (iii) measuring an amount of reporter activity produced.
 37. A compound capable of modulating GPCR activity identified by a method according to claim 33 or claim
 36. 38. A method of determining whether a G-Protein Coupled Receptor (GPCR) is coupled to a cell signalling pathway, the method comprising comparing the ligand-independent reporter system output of a Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising: (i) a G-Protein Coupled Receptor (GPCR)-regulated signalling pathway which is derepressed during the mitotic phase of cell growth; (ii) a reporter system for reporting a signal mediated by the GPCR-regulated signaling pathway; with the reporter system output of a reference cell which lacks a functional GPCR
 39. A Schizosaccharomyces pombe (Sz. pombe) yeast cell comprising: (i) a G-Protein Coupled Receptor (GPCR)-regulated signalling pathway which is derepressed during the mitotic phase of cell growth, wherein the GPCR is absent or otherwise rendered non-functional; (ii) a reporter system for reporting a signal mediated by the GPCR-regulated signing pathway.
 40. An assay kit comprising a yeast cell or isolated nucleic acid molecule as defined in any one of claims 1 to 30 and
 39. 